Compositions and methods for inhibition of mmp:mmp-substrate interactions

ABSTRACT

The present invention provides compounds for disrupting the binding of a matrix metalloprotease (MMP) protein to a substrate protein at an interaction site other than the protease catalytic site. In particular the inventive compounds inhibit the MMP&#39;s ability to cleave a substrate protein. In some cases the compound may prevent activation of transforming growth factor beta (TGFβ). The compounds are preferably polypeptide fragments of the hemopexin-like domain of the MMP, but may be mimetics thereof or peptides or mimetics of the portion of the MMP substrate protein to which the MMP interacts.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority pursuant to 35 U.S.C.§119(e) of U.S. provisional patent application No. 61/391,446, filedOct. 8, 2010, which is hereby incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention is directed to peptides, peptide-like mimeticcompounds, and methods of using same in inhibiting proteolyticinteractions between the hemopexin domain of matrix metalloprotease(MMP) and substrate proteins. In some embodiments, the present inventionmay be used to inhibit activation of Transforming Growth Factor β.

BACKGROUND OF THE INVENTION

Osteoarthritis (OA) is a disease affecting joints following (a)cartilage injury; (b) exposure to excessive load-bearing, or repetitiveuse; or (c) general aging. OA affects 10% of the world's 60 years andolder population. It is characterized by joint pain and dysfunction,degradation of joint cartilages, decreased proteoglycan content inarticular cartilage, production of osteophytes (calcified tissue in themargins of the articular cartilage) and fibrosis of the synovial liningof the joint. The unchecked actions of MMPs and elevated activation oftransforming growth factor beta (TGFβ) have been implicated as a primarycause for osteoarthritic cartilage degradation.

TGFβ biological activity is modulated by protease-mediated activation todisassemble latency complexes. TGFβ is secreted as a small latentcomplex that is covalently associated with latent TGFβ binding protein 1(LTBP1) to form the TGFβ large latent complex. LTBP1 anchors the TGFβlarge latent complex (TGFβ LLC) to the extracellular matrix (ECM). Thiscomplex must be released from the extracellular matrix in order for TGFβto become activated for signaling. TGFβ is secreted as a small latentcomplex in non-covalent association with its N-terminal latencyassociated peptide, β-LAP. β-LAP and LTBP1 have been implicated inproper processing, secretion, and guidance of the TGFβ LLC to the ECMfor storage. Analysis of TGFβ distribution in bone indicates that thebulk of TGFβ is stored in the ECM as a 100 kD TGFJ3 small latent complex(TGFJ3 SLC) and a 270 kD TGFβ LLC. TGFβ is only capable of binding thesignaling receptor complex in its mature, 25 kD, homodimeric form.Therefore, activation must occur through a tightly controlled series ofproteolytic steps. Plasmin, elastase, chymase, thrombospondin, MMP9,MMP3 and MMP13 have all been implicated in activation of TGFβ resultingin release of the mature receptor-binding homodimer. In addition, analternatively spliced short form of LTBP1 can form the large latentcomplex with TGFβ. It has been demonstrated that this form of the TGFβLLC including the short LTBP1 can be more readily removed from theextracellular matrix.

MMP inhibitors, such as batimastat, marimastat, CGS-27023A, andprinomastat, have been used to treat OA. However, those attempts haveresulted in severe side-effects known as MMP-induced musculoskeletalsyndrome which includes joint stiffness, inflammation, and symptomsmanifested as pain in the hands, arms, and shoulders. Therefore, thereremains a need for compounds and methods for inhibiting activation ofTGFβ, and treating OA as well as other TGFβ-associated indicationswithout the adverse effect of MMP-induced musculoskeletal syndrome.

SUMMARY OF THE INVENTION

The presently disclosed novel compounds and methods described herein aidin inhibiting the binding of MMPs to target substrate proteins. In somecases the inventive compounds may aid in preventing cleavage of MMPsubstrate proteins. Substrate proteins include Latent TGFβ BindingProtein 1 (LTBP1), collagen, aggrecan, perlecan and fibronectin.Exemplary MMPs include MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP11, MMP12,MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23B, MMP24,MMP25, MMP27, MMP28, MMP29.

In one embodiment, the compounds and methods may comprise peptidesequences derived from the hemopexin domain of an MMP protein. In someembodiments the compounds and methods are used to inhibit cleavage, byMMP, of Latent TGFβ Binding Protein 1 (LTBP1), preventing the release ofactivated transforming growth factor beta (TGFβ) from the LTBP1 complex.In one particular embodiment the presently described compounds andmethods aid in inhibiting the binding of the hemopexin domain of MMP13to LTBP1.

The disclosed invention provides compounds and methods of using samehaving matrix metalloprotease (MMP) inhibitory activity withoutaffecting the enzyme's catalytic domain. In some embodiments, thepresently disclosed compounds and methods inhibit binding of the MMPprotein to a substrate protein by disrupting binding at non-catalyticsites. In various embodiments, the compounds prevent the binding of MMPproteins and substrate proteins by binding to the substrate protein ator near the MMP biding site. In some embodiments the MMP binding sitemay be a calcium-binding, EGF-like domain.

The present inventors have discovered that certain MMPs function byinteracting with substrate proteins at the MMP hemopexin-like domain. Insome MMP proteins, the hemopexin-like domain and the catalytic domainmay be located at different, physically separated portions of theenzyme. The MMPs include MMP13, MMP14, MMP16, MMP2, MMP9, MMP19, MMP17,MMP15, MMP20, MMP1, MMP24, MMP25, MMP3, NIMP 21, MMP28, MMP8, MMP12,MMP27, MMP11, and MMP10.

In an embodiment, the present invention provides compounds having matrixmetalloprotease 13 (MMP13) inhibitory activity. In particular thecompounds inhibit MMP13's ability to cleave LTBP1, thereby, inhibitingthe activation of TGFβ. Because the disclosed compounds inhibit TGFβactivation, they are useful in combating conditions to which TGFβactivation contributes. Diseases and conditions involving dis-regulationof TGFβ include cartilage degeneration and osteoarthritis (OA).Accordingly, the present invention also provides pharmaceuticalcompositions and methods for treating such conditions.

In another embodiment, the present invention relates to compounds havingMMP13 inhibitory activity and TGFβ activation dysregulation activity. Inone particular embodiment, the compound is a peptide fragment of thehemopexin-like (1PEX) domain of the MMP13 protein. In some embodiments,the compound includes peptide-like or non-peptide compounds. In variousembodiments, the compound may be a mimetic compound designed to mimicthe size, shape, charge, and binding characteristics of the all or aportion of a hemopexin domain or binding surface of a hemopexin domain.

One particular polypeptide sequence of a 1PEX domain of MMP13 isdepicted in SEQ ID NO: 1. Fragments of the 1PEX domain active forinhibiting TGFβ activation can be identified as shown in Example 2below. Bioinformatics is used to identify candidate fragments that caninteract with latent TGFβ binding protein (LTBP 1). Those candidatefragments are then tested for their ability to inhibit TGFβ activation.Preferably, the fragment contains at least 6 amino acids, morepreferably 6 to 50 amino acids, most preferably about 19 amino acids.The preferred fragments of SEQ ID NO: 1, suitable for inhibitingMMP13-LTBP 1 interaction, and subsequent TGFβ activation comprise; aminoacids 17-26; amino acids 93-111; amino acids 101-150; amino acids109-147; amino acids 125-147; amino acids 151-192; amino acids 160-180;amino acids 51-100; amino acids 60-83; amino acids 1-50; amino acids16-50; amino acids 168-186; and amino acids 109-129.

In various embodiments the inventive compound may disrupt aprotein:protein interaction that is between a MMP and a substrateprotein, wherein the interaction is not mediated by the hemopexin domainof MMP and/or the calcium-binding, EGF-like domain of a substrateprotein. Thus, in one embodiment the inventive compound may be derivedfrom an MMP protein but have no homology to a hemopexin domain.

In various embodiments, the inventive peptide may be non-identical tothe peptides fragments of the MMP protein. In some embodiments, theinventive compound may be greater than about 95% identical or similar toa peptide disclosed herein, or greater than about 90%, about 85%, about80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%,about 45% about 40%, about 30%, or about 20% identical or similar to apeptide sequence disclosed herein. For example, where one inventivecompound comprises amino acids 93-101 of SEQ ID NO: 1, other inventivecompounds may be 95% identical to that peptide. In some embodiments thenon-identical amino acids may be natural amino acids, non-natural aminoacids, may be a modified version of the identical amino acid,conservative amino acid substitution, non-conservative amino acidsubstitution, or a small molecule with a property very similar to thatof the non-substituted, non-modified, amino acid. Non-natural andderivatized amino acids are readily available from common suppliers ofchemical reagents, for example, Sigma-Aldrich. In some embodiments, thenative amino acid may be substituted with a small molecule that lacks apeptide backbone.

In yet another embodiment, the present invention relates to methods forinhibiting the activation of TGFβ, by contacting LTBP1 with theinventive compound to prevent cleavage of LTBP1 by a MMP. Preferably,the MMP-LTBP1 interaction disrupted by the inventive compounds isbetween LTBP1 and MMP14, MMP13, MMP9, MMP3 or MMP2.

In a further embodiment, the present invention relates to methods forinhibiting the activation of TGFβ by contacting the MMP with an amountof a compound according to the present invention effective to inhibitthe activation of TGFβ. In some embodiments the inventive compound maycomprise peptide sequences similar to a region of a substrate protein,for example, LTBP1. In further embodiments, the region of the substrateprotein may be a calcium-binding, EGF-like domain.

In a further embodiment, the invention also relates to methods fortreating a mammal suffering from osteoarthritis or cartilagedegeneration by administering to the mammal an amount of a compoundaccording to the invention sufficient to alleviate the effects ofosteoarthritis or cartilage degeneration. In some embodiments theinventive compound may be administered locally or systemically. Invarious local administration embodiments, the administration may be byinjection, patch, cream, lotion, etc. In some embodiments where systemicadministration is appropriate, for example osteoarthritis,administration of the inventive compound may be oral or nasal, forexample, a nasal spray.

In further embodiments, the present invention relates to compounds forinhibiting the interaction of MMP13, MMP14, MMP16, MMP2, MMP9, MMP19,MMP17, MMP15, MMP20, MMP1, MMP24, MMP25, MMP3, MMP21, MMP28, MMP8,MMP12, MMP27, MMP11, and MMP10 and their substrates, without affectingthe catalytic domain. The amino acid sequence for the substrateinteracting, hemopexin-like domain of MMP13 is depicted in SEQ ID NO: 1;MMP14 is depicted in SEQ ID NO: 2; MMP16 is depicted in SEQ ID NO: 3;MMP2 is depicted in SEQ ID NO: 4; MMP9 is depicted in SEQ ID NO: 5;MMP19 is depicted in SEQ ID NO: 6; MMP17 is depicted in SEQ ID NO: 7;MMP15 is depicted in SEQ ID NO: 8; MMP20 is depicted in SEQ ID NO: 9;MMP1 is depicted in SEQ ID NO: 10; MMP24 is depicted in SEQ ID NO: 11;MMP25 is depicted in SEQ ID NO: 12; MMP3 is depicted in SEQ ID NO: 13;MMP21 is depicted in SEQ ID NO: 14; MMP28 is depicted in SEQ ID NO: 15;MMP8 is depicted in SEQ ID NO: 16; MMP12 is depicted in SEQ ID NO: 17;MMP27 is depicted in SEQ ID NO: 18; MMP11 is depicted in SEQ ID NO: 19;and MMP10 is depicted in SEQ ID NO: 20.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing a model of MMP13 release of TGFβ from thelarge latent complex with LTBP1.

FIG. 2 shows that hypertrophic chondrocytes produce a novel large latentTGFβ complex. (A) Day 19 embryonic chick tibial growth plates wereextracted, immunoprecipitated with antiserum to LTBP-1 or MMP13 andimmunoblotted with antiserum to TGFβ. Lane 1, immunoprecipitated withLTBP-1 antiserum; Lane 2 immunoprecipitated with MMP13 antiserum; Lane 3immunoprecipitated with pre-immune serum IgG. LLC=TGFβ large latentcomplex; LAP=TGFβ latency associated peptide; TGFβ=active TGFβ homodimer(B) Day 14 rat newborn tibial growth plate cartilage wasimmunoprecipitated with antiserum to TGFβ and immunoblotted withantiserum to MMP13. Soluble LLC=soluble form of the TGFβ large latentcomplex; Inactive 13=prozymogen form of MMP13; Active 13=activated formof MMP13. (C) Conditioned media from day 5, serum-free, latehypertrophic chondrocyte cultures was incubated with biotin-labeled,TGFβ polyclonal antibody, passed over a strep-avidin magnetic beadcolumn and immunounoblotted with either NIMP13 or LTBP-1 antibodies asdescribed in Materials and Methods. Lefthand lanes=flow through;Righthand lanes=eluted protein.

FIG. 3 shows that hypertrophic chondrocytes produce the short form ofLTBP-1. Total RNA was isolated from day 5 alginate early hypertrophic(EH) and late hypertrophic (LH) chondrocytes. Densitometric values arenormalized to 18 s rRNA from a sample n>5 separate cultured experiments.Graphs and statistics generated using Prism GraphPad version 3.03. (A)expression of markers of hypertrophy and (B) expression of thecomponents of the TGFβ LLC.

FIG. 4 shows that hypertrophic chondrocytes produce the components ofthe TGFβ LLC. Early (EH) and late (LH) hypertrophic chondrocytes werereared in serum-free alginate culture. At day 5, 20 μg equal totalprotein of conditioned media (soluble), cell-associated matrix(territorial matrix) and CHAPS-extracted cell pellet (cell layer) wereelectrophoresed under reduced conditions as described in Materials andMethods. (A) TGFβ 2 polyclonal antibody; (B) MMP-13 polyclonal antibody;and (C) LTBP-1 polyclonal antibody. LLC=TGFβ large latent complex;Sol=soluble TGFβ LLC; SLC=TGFβ small latent complex; β-LAP=TGFβ latencyassociated peptide and H=TGFβ homodimer.

FIG. 5 shows the detection of collagen type X in cell-free matrices.Late hypertrophic chondrocyte cell-free matrices were prepared,immunolabeled with collagen type X polyclonal antibody (red) and nucleicounterstained with Hoechst dye (blue) as described in Materials andMethods. Images were captured with a 100× objective and colorcompositions constructed using ImagePro software as described inMaterials and Methods. (A) cell-free extracellular matrix; (B) cellcytoplasm; (C) secondary antibody control; (D) three-dimensional imagecapture at 45° of rotation.

FIG. 6 shows TGFβ2, MMP-13, and LTBP-1 localize to both theextracellular matrix and cytoplasm. Two-dimensional analysis of latehypertrophic chondrocytes labeled with polyclonal antibodies: (A)TGF-β2; (B) MMP-13; and (C) LTBP-1. Three-dimensional analysis depictedevery 45° of rotation: (D) TGFβ 2; (E) MMP-13; and (F) LTBP-1. Nucleiare counterstained with Hoechst dye (blue). Arrows in panels C and Findicate extracellular matrix staining.

FIG. 7 shows co-localization of MMP-13 and LTBP-1 produced by latehypertrophic chondrocytes. Hypertrophic chondrocytes were double-labeledwith MMP-13 (red) and LTBP-1 (green) and nuclei counter-stained (blue)as described in Materials and Methods. Co-localization of the proteinsappears as a yellow to orange color. (Al-A3) MMP-13 polyclonal antibody,(B1-B3) LTBP-1 polyclonal antibody, and (C1-C3) co-localized image ofthe same field. Co-localization within ECM (arrow, panel C3) andcytoplasm (arrow, panel C1) is observed. (D) Three-dimensional analysisof co-localized MMP-13 and LTBP-1 demonstrating staining within theextracellular matrix and cytoplasm.

FIG. 8 shows three-dimensional modeling of MMP13 and LTBP1 non-covalentinteraction. (A) MMP13 and LTBP1 short (870 AA toward the C-terminal);Pink: Helices, Yellow: Beta sheets and White: coils. N: Amino terminal.C: Carboxyl terminal. (B) Three-dimensional model of MMP13-LTBP1interaction; MMP13:green, LTBP1: blue. LLC=large latent complex;H=hemopexin domain; CE=EGF-Ca domain; C=cysteine; E=EGF-like domain;P=N-terminal catalytic domain of MMP13; Linker region=protease-sensitivehinge region of LTBP. (C) Protein-protein interface prediction ofMMP13-LTBP1 complex shown in B. Red: Highest scoring patch (probablebinding site). Yellow: Second highest scoring patch. Green: thirdhighest scoring patch.

FIG. 9 shows binding of MMP13-derived peptides with cartilage tissueextracts. Cartilage from day 17 avian embryo sterna was extracted with0.5% CHAPS buffer and binding assays conducted. Scr=scrambled control;+cold=unlabeled competition. *=p<0.001 compared to scrambled.***=p<0.001 compared to MMP13-derived peptide by ANOVA with Tukey'sAnalysis. (A) Resting cartilage and MMP13-19 peptide. (B) Hypertrophiccartilage and MMP13-19 peptide. (C) Hypertrophic cartilage and MMP13-10peptide D. hypertrophic cartilage and MMP13-6 peptide.

FIG. 10 shows binding of MMP13-derived peptides with isolatedextracellular matrix. Binding assays utilizing intact hypertrophicchondrocyte-produced extracellular matrix, LTBP1 immunoprecipitates ofhypertrophic chondrocyte-produced extracellular matrix and recombinantprotein of the calcium-binding, EGF-like domains of LTBP1 (30) wereperformed as described in Materials and Methods. *=p<0.001 compared toscrambled. ***=p<0.001 compared to MMP13-derived peptide by ANOVA withTukey's analysis. (A) Intact hypertrophic chondrocyte-producedextracellular matrix and MMP13-19 or MMP13-10 peptide binding. (B) 25 μgeluates of hypertrophic cartilage immunoprecipitated with polyclonalantibody to LTBP1 and MMP13-19 peptide binding. (C) 15 ng of recombinantprotein of the calcium-binding, EGF-like domains and MMP13-19 orMMP13-10 or MMP13-6 peptide binding.

FIG. 11 shows MMP13-19 peptide effects on endogenous activation of TGFβproduced by hypertrophic chondrocytes. Primary sternal chondrocytes fromday 17 avian embryos were isolated and plated in alginate culture (2).At day 5 in serum-free culture, 10 nM, 100 nM or 250 nM MMP13 peptidewas added to the cultures for 24 hours. Conditioned media was collectedand concentrated with Centricon-10 spin filters (Fisher Scientific) andan ELISA (R&D Systems) performed to measure total TGF□ produced versusendogenously activated TGF□ produced. The graph shows endogenouslyactivated TGF□ as percentage of the total TGF□ produced. Statisticalanalysis was calculated by ANOVA with Tukey's test utilizing PrismGraphPad software. n>5 separate culture experiments.

FIG. 12 is a drawing showing the 3-D docking interaction of MMP13, MMP2,and MMP9 with LTBP1. Amino acid sequences of proteins analyzed wereobtained from Swiss-Prot data base (http://www.ebi.ac.uk/swissprot/).Pfam data base (http://www.sanger.ac.uk/Software/Pfam/) was used forprotein domain analysis. Pfam-A is based on hidden Markox model (HMM)searches, as provided by the HMMER2 package (http://hmmer.janelia.org/).In HMMER2, like BLAST, E-values (expectation values) are calculated. TheE-value is the number of hits that would be expected to have a scoreequal or better than this by chance alone. A good E-value is muchsmaller than 1 because 1 is what is expected that sequences are similarby chance. In principle, the significance of a match is predicated on alow E-value. 3D models were generated using I-TASSER database(http://zhang.bioinformatics.ku.edu/I-TASSER/). Protein docking modelswere generated using Vakser lab database(http://vakser.bioinformatics.ku.edu/resources/gramm/grammx/).Protein-protein interface prediction data was generated using PIP-Preddatabase (http://bioinformatics.leeds.ac.uk/ppi_pred/index.html). Imageswere generated using Jmol software (http://jmol.sourceforge.net/).

FIG. 13: Three dimension model of dog mmp13.

FIG. 14: Collagen triple helix.

FIG. 15: dog MMP13-collagen complex.

FIG. 16: MMP13-derived modifying compound (MC1).

FIG. 17: MMP13-collagen-MC1 complex.

FIG. 18: MC1-aggrecan complex.

FIG. 19: MC2-Fibronectin III complex.

FIG. 20; List of various embodiments of the present inventive compoundsfor interfering with interactions of various MMPs and their substrates

FIG. 21, shows digestion kinetics of MMP13 catalytic domain on hingesubstrate 21A and scrambled substrate, 21B. EDAN-DABSYLfluorescence-labeled REHARGS peptide, representing theprotease-sensitive hinge region of LTBP1, was assayed with MMP13catalytic domain (Enzo Laboratories). Enzyme kinetics were calculated byMichaelis-Menten and non-linear regression software available with thePrism GraphPad program.

-   -   A.) MMP13 enzymatic activity on the REHARGS peptide.    -   B.) MMP13 enzymatic activity on the REHARGS peptide versus a        scrambled control sequence, AREHGSR

FIG. 22A is a bar graph showing enzymatic activity of various cartilageextracts, and 22B is a bar graph comparing MMP13 to various extracts.CHAPS buffer extracts of avian sterna cartilage were assayed with thefluorescence labeled REHARGS substrate (H) versus a scrambled controlsequence (S) as described in the legend for FIG. 5. Extracts of latehypertrophic, early hypertrophic and resting cartilage were assayed.

-   -   A.) MMP13 catalytic domain and the three cartilages were assayed        with Hinge and Scrambled peptide. ANOVA with Tukey's test were        performed with Prism GraphPad software.    -   B.) ANOVA with Tukey's test were performed to compare Hinge        peptide activity in the different conditions.

FIG. 23 shows histopathology slides of chronic treatment of anMIA-induced OA. rat model MIA (3 mg in 50 ul) will be injected into thecapsule of the stifle through the infrapatellar ligament of the rightknee (Janusz, M. J., Hookfin, E. B., Heitmeyer, S. A. et al. (2001)Osteoarthritis Cartilage 9, 751-760; Guingamp, C., Gegout-Pottie, P.,Philippe, L., Terlain, B., Netter, P., and Gillet, P. (1997) ArthritisRheum. 40, 1670-1679) The contralateral knee, injected with saline, willserve as control for the experiment. Disease parameters are clearlymeasurable within three to four weeks following injection. Animals wereinjected bi-weekly (weeks 4-12) with various doses of MMP13-19 peptide.Saline and BMP-7 (50 uM) included as negative and positive controls,respectively. Animals were sacrificed, joints dissected, fixed informalin, decalcified, embedded in paraffin and sectioned. Samples werestained with H&E or saffranin O. Grading of OA pathology followed theMankin Scale (Pritzker, K. P., Gay, S., Jimenez, S. A., Ostergaard, K.,Pelletier, J. P., Revell, P. A., Salter, D., and van den Berg, W. B.(2006) Osteoarthritis Cartilage 14, 13-29).

FIG. 24 shows safranin-O stained histopathology and grading of acutetreatment of a positive control in an MIA-induced model of OA. OA wasinduced as described in FIG. 23. Animals were injected weekly (weeks1-4) with various doses of MMP13-19 peptide. Saline and BMP-7 (50 uM)included as negative and positive controls, respectively. Animals weresacrificed, joints dissected, fixed in formalin, decalcified, embeddedin paraffin and sectioned. Samples were stained with H&E or saffranin O.Grading of OA pathology followed the Mankin Scale (Pritzker, K. P., Gay,S., Jimenez, S. A., Ostergaard, K., Pelletier, J. P., Revell, P. A.,Salter, D., and van den Berg, W. B. (2006) Osteoarthritis Cartilage 14,13-29).

FIG. 25 shows hematoxaylin and eosin stained histopathology and gradingof acute treatment positive control in osteoarthritis model.

FIG. 26 shows safranin-O stained histopathology and grading of positivetreatment control (50 uM BMP7) in osteoarthritis model.

FIG. 27 shows hematoxaylin and eosin stained histopathology and gradingof positive treatment control (50 uM BMP7) in osteoarthritis model.

FIG. 28 shows safranin-O stained histopathology and grading of low dosetreatment (250 nM MMP13-19 peptide) in osteoarthritis model.

FIG. 29 shows hematoxaylin and eosin stained histopathology and gradingof low dose treatment (250 nM MMP13-19 peptide) in osteoarthritis model.

FIG. 30 shows safranin-O stained histopathology and grading of high dosetreatment (2.5 uM MMP13-19 peptide) in osteoarthritis model.

FIG. 31 shows hematoxaylin and eosin stained histopathology and gradingof high dose treatment (2.5 uM MMP13-19 peptide) in osteoarthritismodel.

FIG. 32 shows safranin-O, and hematoxaylin and eosin stained mid jointhistopathology of low dose peptide inhibitor treatment in osteoarthritismodel.

FIG. 33 shows safranin-O, and hematoxaylin and eosin stained mid jointhistopathology of saline and BMP7 treatment in osteoarthritis model.

FIG. 34 shows safranin-O, and hematoxaylin and eosin stained mid jointhistopathology comparing BMP7 treatment and high dose peptide inhibitortreatment in osteoarthritis model.

FIG. 35 shows micro computer tomography (micro CT) and table of totaland bone volume in patellar and total joint 21 days post OA inducementwith and without peptide treatment. Isolated joints were analyzed byMicro CT to measure cortical bone, trabecular bone and cartilage of thepatella, femur and tibia, the production of chondrophytes and tissuemineralization in response to treatment. Total mineralization in thepatella, femur and tibial cartilages, as well as subchondral bone, werecalculated with Scanco μCT software. Micro-CT was conducted with aScanco uCT 35 (Scanco Medical, Bassersdorf, Switzerland) system. Scansof 15 μm voxel size, 55 KVp, 0.36 degrees rotation step (180 degreesangular range) and a 600 ms exposure per view produced from jointsimmersed in phosphate buffered saline.

FIG. 36 is a bar graph showing femoral-tibial head joint space for 4week acute treatment Osteoarthritis control, normal, and peptide treatedanimals. The joint were X-rayed to measure joint space changes as anindicator of the progression of OA (Messent, E. A., Ward, R. J., Tonkin,C. J., and Buckland-Wright, C. (2005) Osteoarthritis Cartilage 13,463-470).

FIG. 37 is a bar graph showing micro CT calculated ratio of bone volumeto total volume for 4 week acute Osteoarthritis control, normal, andpeptide treated animals.

FIG. 38 is a bar graph showing average stride for 10 week and 12 weekOsteoarthritis control, normal, and peptide treated animals. A stridetest was administered weekly during the course of treatment to determinefunctional mobility in the animals. Briefly, rat's hind paws were inked,the animals timed while they walked a short path and the distancebetween hind leg strides measured (Hruska, R. E., Kennedy, S., andSilbergeld, E. K. (1979) Life Sci. 25, 171-179)

FIG. 39 shows details cytotoxicity data for treatment in vitro withMMP13-19 peptide. Primary chondrocytes from early and late hypertrophicstage were cultured from Day 17 avian upper sternum. Late hypertrophicchondrocytes were isolated from the core region of the avian sterna.Following 3-4 hours collagenase and trypsin digestion, cells werecentrifuged and filtered through 0.45 um Nitex filter. Isolated cellswere resuspended in 1.2% alginate and forced into beaded structures with102 mM CaCl2 and rinsed in 0.15M NaCl for a final density of 5×106cells/ml. Alginate bead cultures were covered in 2 mls complete serumfree DMEM high glucose media including 1 mM cysteine, 1 mM sodiumpyruvate, 2 mM L-glutamine, 50 μg/ml penicillin/streptomycin. L-ascorbicacid was added to the culture at 30 ug/ml on day 2 and 50 ug/ml on day5. Cytotoxicity was assessed by Alomar Blue Assay (Invitrogen) onprimary chondrocytes and a monocyte cell line incubated for 24 hourswith PxTx001-1. Absorbance was recorded at 570 nm for every hour up to24 hrs to monitor both proliferation and metabolic activity.

FIG. 40 is a bar graph showing mRNA expression in chondrocytes treatedwith MMP13-19 or a commercially available MMP inhibitor. Time coursetreatment was performed at 6, 12 or 24 hours with 10 nM, 100 nM, 250 nMPxTx001-1 or 6.5 uM commercially available MMP13 specific inhibitor(Calbiochem). Following a quick dissolution in 0.5M EDTA to releasecells from alginate cultures, total RNA was isolated through Trizolmethod and reverse-transcribed via SuperScript First-Strand SynthesisSystem (Invitrogen). cDNA samples were subjected to QuantiTech SyBrGreen(Qiagen) real time PCR. Samples were loaded into a 96 well plate intriplicate as 1 ul or 2 ul cDNA for each condition and primersrespectively. Expression of markers of chondrocyte maturation (collagentype X, MMP13 and alkaline phosphatase) was compared to an internalstandard of 18srRNA using ABI Prism 7000 sequence detection system(Applied Biosystems). Fold difference compared to untreated cultures wasgraphed using Prism Graph Pad and statistical analysis of one-way ANOVAand standard error of the mean were calculated with associated software.

FIG. 41 is a matrix showing conservation of amino acids in the MMP13-19inhibitory peptide throughout the MMP family of proteins.

FIG. 42 shows binding of BMP2 with MMP7 (42A) or MMP12 (42B).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Described herein are compounds for inhibiting matrix metalloprotease(MMP)-substrate protein-protein interactions. The present inventivecompounds are designed using high definition modeling of theMMP-substrate binding interface. In some embodiments, inhibition doesnot affect protease activity. In various embodiments, the inventivecompounds are fragments of the hemopexin-like domain of a MMP orfragments of an MMP binding protein. In some embodiments, the inventivecompound may be a an engineered peptide, peptide derivative, or othermolecule comprising a chemical and three-dimensional structure designedto bind either MMP or the MMP-substrate at the binding interface.

In various embodiments, the inventive peptide, peptide derivative, orother peptide-like molecule may be identical to a peptide fragment of aMMP protein. In various other embodiments the inventive compound may benon-identical to a peptide fragment of MMP. In various embodiments theinventive compound may include non-natural amino acids, derivatizednatural amino acids, conservative substitution, non-conservativesubstitutions, or combinations thereof. In various embodiments theinventive compound may be greater than about 95%, about 90%, about 85%,about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about50%, about 45%, about 40%, about 30%, or about 20% identical or similarto a peptide sequence disclosed herein. One of skill in the art ofpeptide synthesis may use various programs and algorithms to determinehomology among proteins and peptides. In various embodiments, homologymay be determined by a weighted system that counts non-conservativesubstitutions at a specific position differently than conservativesubstitutions (based on charge, hydrophobicity, size, etc). On suchweighting algorithm may be found at homology database serverhttp://www.clustal.org/clustal2/, among others.

One of skill in the art of peptide synthesis may choose to incorporatenon-natural amino acids, derivatized amino acids, or small molecules toaid in preventing or reducing degradation of the inventive compound.Design of ligands and small molecules, such as is described by Kubinyi,is well known in the art (Structure-based design of enzyme inhibitorsand receptor ligands, Curr. Op. in Drug Disc. and Development, 1998 Vol1 No 1; incorporated in its entirety by express reference).

In some embodiments, the compound may be referred to as a mimetic. Amimetic is a protein-like chain designed to mimic the pharmacologicalactivity and the structure of a peptide. Mimetics typically arise frommodification of an existing peptide in order to alter the molecule'sproperties. For example, they may arise from modifications (such asusing unnatural amino acids, conformational restraints, etc.) to changethe molecule's stability or biological activity. Preferably, mimetic, asused herein, means a molecule that is designed to resemble thehemopexin-like fragment and to function as a MMP inhibitor. Because thehemopexin like domain is spaced apart from the catalytic domain on theMMP, the compound inhibits the MMP by interfering with the interactionof the hemopexin-like domain with the substrate. That way, the catalyticdomain is not affected.

MMPs include MMP13, MMP14, MMP16, MMP2, MMP9, MMP19, MMP17, MMP15,MMP20, MMP1, MMP24, MMP25, MMP3, MMP21, MMP28, MMP8, MMP12, MMP27,MMP11, and MMP10.

In some embodiments, the inventive compound is a fragment of thehemopexin-like domain of the MMP or mimetics thereof. The polypeptidesequence of hemopexin-like domains for MMP13, MMP14, MMP16, MMP2, MMP9,MMP19, MMP17, MMP15, MMP20, MMP1, MMP24, MMP25, MMP3, MMP21, MMP28,MMP8, MMP12, MMP27, MMP11, and MMP10 are depicted in SEQ. ID NOS: 1-20,respectively. The fragments appropriate for the present invention can bedetermined using the methods of the Examples below. Bioinformatics isused to select candidate fragments in the hemopexin-like domain of theMMP that can potentially interact with its substrate (e.g., LTBP-1).Those candidate fragments are then tested for their ability to inhibitactivation of the substrate by the MMP. Preferably, the fragmentcontains at least 6 amino acids, more preferably 6 to 50 amino acids,and most preferably 19 amino acids.

The present invention provides compounds and methods for disruptinginteractions between matrix metalloprotease (MMP) and a variety ofsubstrate target proteins that interact with MMP through a MMP hemopexindomain. Substrate target proteins include LTBP1, aggrecan, fibronectins,and collagens.

Compounds of the present invention for inhibiting various MMPs arepresented in FIG. 20.

The compounds of the present invention can also encompass otherfragments of the MMP which can be determined by first generating a threedimension (3D) structure(s) for the molecules of interest (the MMP andits substrate), preferably by computer Modeling. The modeling can bedone using software well-known and available in the art, such as thePPI-Pred database from Leads University in the United Kingdom. From the3D structure, the interacting portions of the hemopexin-like domain ofthe MMP and the substrate are determined. Branching or non-branchingpeptides may block the interaction between the MMP and its substrateprotein. Inventive compounds, including peptides and peptide mimetics,may be designed from analysis of the sequences of the two proteins atthe protein-protein interlace. The protein-protein interface maydescribe the surfaces of the MMP and substrate protein that may interactnon-covalently.

Designed compounds and peptides may be tested for their ability toinhibit the MMP-substrate interaction. In some embodiments, theinventive compound may compete with an MMP or its substrate protein forbinding.

In some embodiments, the best performing peptides are selected based onin-vitro evaluation-of their ability to inhibit the interaction ofinterest. For example, if inhibition of TGFβ release is desired, thepeptide performance is based on its ability to inhibit TGFβ release intissue culture system as described in the Examples below. In someembodiments, the “best performing peptide” may be based on its bindingaffinity for its target, which may be MMP or a substrate protein. Insome embodiments the “best performing peptide” may be the compound whichhas the greatest resistance to degradation.

In the case of MMP9, for example, a peptide may be chosen for itsability to prevent homodimerization. In that case, the peptides areevaluated by examining tissue cultures treated with the peptides by,e.g., western blot for MMP9. In-vitro evaluation techniques are apparentto one skilled in the art depending on the desired MMP and substrateinteraction.

In various embodiments of the inventive compound, the compound maydisrupt an MMP's ability to bind a substrate protein other than LTBP1.For example, the inventive compound may prevent the biding of MMP13 tocollagen type II. In some embodiments, the inventive compounds may beuseful in combating conditions such as Matrix degeneration. Accordingly,the present invention also provides pharmaceutical compositions andmethods for treating such conditions.

In many embodiments, the inventive compound may be similar to a humanMMP protein or a human MMP substrate protein. In other embodiments, theinventive compound may be based upon a non-human MMP protein or MMPsubstrate protein. In some embodiments, for example, the inventivecompound may be homologous to a dog protein, for example dog MMP13. Theinventive compound may also be homologous to a dog MMP substrateprotein.

In various embodiments the inventive compound may disrupt aprotein:protein interaction that is between a MMP and a substrateprotein, wherein the interaction is not mediated by the hemopexin domainof MMP and/or the calcium-binding, EGF-like domain of a substrateprotein. Thus, in one embodiment the inventive compound may be derivedfrom an MMP protein but have no homology to a hemopexin domain. Forexample, interactions between MMP7 and BPM2 or MMP12 and BMP2 (FIG. 42),which may be involved in fracture healing and/or fracture nonunion,

In some embodiments, In some embodiments, interface data based upon MMP7or 12 interacting with BMP2 indicates low energy needed for theinteraction (average −10.7 kcal/mol), and a large surface area (Average967.5 kcal/mol). This data indicates strong binding due to hydrogenbonds and disulfide bonds formations, and may help to explain previouslypublished data (Fajardo et al., Matrix metalloproteinases that associatewith and cleave bone morphogenetic protein-2 in vitro are elevated inhypertrophic fracture nonunion tissue. J Orthop Trauma. 2010 September;24(9):557-63; incorporated herein by reference in its entirety). MMP7and MMP12 are modeled to interface with different regions of BMP2. Thesedata suggest that at least these two MMPs are not competing andpotentially have synergistic effect on BMP2. The negative effect of MMPson BMP2 function might be through degradation (enzymatic activity orthrough interfering with dimerization of the BMP protein).

MMP may interact with a variety of substrate proteins. For example, MMPsmay interact with For example, For example, MMPs may interact with Forexample, the substrate protein may be selected from among the groupconsisting of MCP-1, MCP-2, MCP-3, MCP-4, Stromal cell-derived factor(SDF), Pro-1L-1β, Pro-IL-8, IL-1-β, IGF-BP, IGF-BP-2, IGF-BP-3,Perlecan, Pro-TNF-α, Pro-MMP-1, Pro-MMP-2, Pro-MMP-2, Pro-MMP-3,Pro-MMP-7, Pro-MMP-8, Pro-MMP-9, Pro-MMP-10, Pro-MMP-13, α1-proteinaseinhibitor, α1-antichymotrypsin, α2-macroglobulin, L-selection,Pro-TGF-β1, Pro-IL-1β, IGFBP-3, IGFBP-5, FGFR-1, Big endothelin-1,Pregnancy zone protein, Substance P, Decorin, Galectin-3,CTAP-III/NAP-2, GROα, PF-4, Cell-surface IL-2Rα, Plasminogen,Pro-a-definsin, Cell surface bound Fas-L, E-cadherin; β4 integrin,Pro-a-definsin, Cell-surface CD44, Cell-surface BOUND tissuetransglutaminase (tTG), 1-selecting, Pro-HB-EGF, e-Cadherin. In manyembodiments the MMP substrate protein may, or may not, have a hemopexindomain. In various embodiments, the MMP may interact with a substrateprotein by binding the substrate with its hemopexin domain. In otherembodiments the interaction between MMP and the substrate protein may bethrough a domain other than the MMP hemopexin domain.

In some embodiments, the inventive compound may comprise modificationsthat may be present on the parent MMP or MMP substrate, which thecompound is based upon. For example, the MMP or MMP-substrate mayinclude glycosylation and phosphorylation within the regioncorresponding to the inventive compound. Thus, bioinformatic tools maybe used to predict these biochemical modification, and the inventivecompounds may be modified to match parent protein features. Thesemodifications may be accomplished by derivativization of the amino acid,or by treating the inventive compound with the appropriate modifyingenzyme, for example a kinase in the case of phosphorylation.

In many embodiments, the inventive compound may be modified to increasethe thermodynamic stability of the inventive compound. In embodimentswhere greater thermodynamic stability is desired, one of skill in theart may choose among many available modifications. For example, one ofskill in the art may engineer di-sulfide bonds within the inventivecompound to increase stability. In other embodiments, the hydrophobiccore of the inventive compound may be engineered to be more stable, orsecondary or tertiary interactions may be engineered or modified toincrease thermodynamic stability.

In various embodiments, the inventive compound may be modified to aid inpreventing, reducing, or inhibiting degradation of the inventivecompound. For example, degradative stability may be enhanced by terminalmodifications, such as acetylation and/or amidation. Other modificationswhich may prevent, reduce, or inhibit degradation of the inventivecompounds may include PEG-ylation and/or use of modified amino acids.

In various embodiments, the inventive compound may be linked to otherheterologous peptides or proteins to enhance resistance to degradationor enhance targetting of the inventive compound to a particular tissueor matrix. For example, in some embodiments the inventive compound maybe linked to a protein which may have affinity for a protein or moleculepresent at or near the site where the inventive compound is needed. Forexample, the inventive compound may be linked to a protein which bindsto hyaluronic acid, which may be present within the matrix or tissuewhere the compound is needed. In some embodiments the linkage may becovalent or non-covalent.

In some embodiments, the inventive compounds may be linked to peptidesor proteins that may, in turn, bid specifically to a carrier in apharmaceutical composition. For example, inventive compounds may includecellulose binding domains, which may be designed to interact with amethylcellulose carrier. Binding to a carrier molecule may aid inprolonging the half-life of an inventive compound.

In an embodiment, the present invention may provide inventive compoundshaving multiple interfacing domains. In many cases protein-proteininteraction involves multiple domains. For efficient modifications,branching molecules might be needed. Bioinformatic tools will be used todetermine appropriate spacing and orientations to achieve the bestdesign.

In an embodiment, the present invention provides compounds that havehybrid structure of peptide and small molecules. In these compounds, thepeptide portion of the compound will provide specificity while the smallmolecule will provide function modification roles.

In another embodiment, the present invention provides methods fortreating a dog suffering from osteoarthritis or cartilage degenerationby administering to the dog a compound of the invention in an amountsufficient to alleviate the effects of osteoarthritis or cartilagedegeneration (100-10,000 microgram). Preferably, the compound isadministered directly to the cartilage of the dog, for example, byinjection.

The present inventors have discovered a mechanism for the activation ofTGFβ by MMP, preferably MMP14, MMP13, MMP9, MMP3, or MMP2. A schematicof the mechanism for MMP13 is shown in FIG. 8. The activation of TGFβ istriggered by the non-covalent interaction of the 1PEX domain of MMP13with the LTBP-1 (particularly the calcium-EGF-like domains of LTBP-1) ofthe TGFβ large latent complex (TGFβ LLC). Once non-covalently associatedwith the TGFβ LLC, the catalytic domain of MMP13 comes into proximity ofand cleaves the LTBP-1 protease-sensitive hinge region, therebyreleasing a soluble form of TGFβ LLC. That soluble form of TGFβ LLC ismore susceptible to proteolytic release of the TGFβ SLC and subsequentactivation of the biologically active TGFβ homodimer. The compounds ofthe present invention are designed to compete with MMP13 for theassociation with the LTBP-1, and thereby preventing the activation ofTGFβ.

It is well known in the art that some modifications and changes can bemade in the structure of a peptide, such as those described herein,without substantially altering the characteristics of that peptide, andstill obtain a biologically equivalent peptide. In one aspect of theinvention, inventive compounds, peptides, and mimetics of the peptidesdescribed here may include peptides, compounds, and mimetics that differfrom the peptide sequences disclosed herein (especially SEQ ID NOs:35-123), by conservative amino acid substitutions. As used herein, theterm “conserved amino acid substitutions” refers to the substitution ofone amino acid for another at a given location in the protein, where thesubstitution can be made without substantial loss of the relevantfunction. In making such changes, substitutions of like amino acidresidues can be made on the basis of relative similarity of side-chainsubstituents, for example, their size, charge, hydrophobicity,hydrophilicity, and the like, and such substitutions may be assayed fortheir effect on the function of the protein by routine testing.

In some embodiments, conserved amino acid substitutions may be madewhere an amino acid residue is substituted for another having a similarhydrophilicity value (e.g., within a value of plus or minus 2.0), wherethe following may be an amino acid having a hydropathic index of about−1.6 such as Tyr (−1.3) or Pro (−1.6)s are assigned to amino acidresidues (as detailed in U.S. Pat. No. 4,554,101, incorporated herein byreference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3);Asn (+0.2); Gln (+0.2); Gly (0); Pro (−0.5); Thr (−0.4); Ala (−0.5); His(−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile (−1.8); Tyr(−2.3); Phe (−2.5); and Trp (−3.4).

In alternative embodiments, conserved amino acid substitutions may bemade where an amino acid residue is substituted for another having asimilar hydropathic index (e.g., within a value of plus or minus 2.0).In such embodiments, each amino acid residue may be assigned ahydropathic index on the basis of its hydrophobicity and chargecharacteristics, as follows: lie (+4.5); Val (+4.2); Leu (+3.8); Phe(+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser(−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glu (−3.5); Gln(−3.5); Asp (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5).

In alternative embodiments, conserved amino acid substitutions may bemade where an amino acid residue is substituted for another in the sameclass, where the amino acids are divided into non-polar, acidic, basicand neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe,Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly,Ser, Thr, Cys, Asn, Gln, Tyr.

In alternative embodiments, conservative amino acid changes includechanges based on considerations of hydrophilicity or hydrophobicity,size or volume, or charge. Amino acids can be generally characterized ashydrophobic or hydrophilic, depending primarily on the properties of theamino acid side chain. A hydrophobic amino acid exhibits ahydrophobicity of greater than zero, and a hydrophilic amino acidexhibits a hydrophilicity of less than zero, based on the normalizedconsensus hydrophobicity scale of Eisenberg et al. (J. Mol. Bio.179:125-142, 184). Genetically encoded hydrophobic amino acids includeGly, Ala, Phe, Val, Leu, lie, Pro, Met and Trp, and genetically encodedhydrophilic amino acids include Thr, H is, Glu, Gln, Asp, Arg, Ser, andLys. Non-genetically encoded hydrophobic amino acids includet-butylalanine, while non-genetically encoded hydrophilic amino acidsinclude citmlline and homocysteine.

Hydrophobic or hydrophilic amino acids can be further subdivided basedon the characteristics of their side chains. For example, an aromaticamino acid is a hydrophobic amino acid with a side chain containing atleast one aromatic or heteroaromatic ring, which may contain one or moresubstituents such as —OH, SH, —CN, —F, —C1, —Br, —I, —NO2, —NO, —NH2,—NHR, —NRR, —C(O)R, —C(O)OH, —C(O)OR, —C(0)NH2, —C(O)NHR, —C(O)NRR,etc., where R is independently (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl,(C₀-C₆) alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl,substituted (C₀-C₆) alkynyl, (C₅-C₂₀) aryl, substituted (C₅-C₂₀) aryl,(C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 memberedheteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl or substituted 6-26 membered alkheteroaryl. Geneticallyencoded aromatic amino acids include Phe, Tyr, and Tryp.

An apolar amino acid is a hydrophobic amino acid with a side chain thatis uncharged at physiological pH and which has bonds in which a pair ofelectrons shared in common by two atoms is generally held equally byeach of the two atoms (i.e., the side chain is not polar). Geneticallyencoded apolar amino acids include Gly, Leu, Val, Ile, Ala, and Met.Apolar amino acids can be further subdivided to include aliphatic aminoacids, which is a hydrophobic amino acid having an aliphatic hydrocarbonside chain. Genetically encoded aliphatic amino acids include Ala, Leu,Val, and Ile.

A polar amino acid is a hydrophilic amino acid with a side chain that isuncharged at physiological pH, but which has one bond in which the pairof electrons shared in common by two atoms is held more closely by oneof the atoms. Genetically encoded polar amino acids include Ser, Thr,Asn, and Gin.

An acidic amino acid is a hydrophilic amino acid with a side chain pKavalue of less than 7. Acidic amino acids typically have negativelycharged side chains at physiological pH due to loss of a hydrogen ion.Genetically encoded acidic amino acids include Asp and Glu. A basicamino acid is a hydrophilic amino acid with a side chain pKa value ofgreater than 7. Basic amino acids typically have positively charged sidechains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include Arg, Lys, and His.

Conservative amino acid changes involve substitution of one type ofamino acid for the same type of amino acid. For example, where charge isbeing conserved, changing Lysine to Arginine is a conservative change,whereas changing Lysine to Glutamic acid is non-conservative. Where sizeis being conserved, a change from Glutamic acid to Glutamine may beconservative, while a change from Glutamic acid to Glycine may benon-conservative

It will be appreciated by one skilled in the art that the aboveclassifications are not absolute and that an amino acid may beclassified in more than one category. In addition, amino acids can beclassified based on known behaviour and or characteristic chemical,physical, or biological properties based on specified assays or ascompared with previously identified amino acids.

Thus an identical sequence will have the same order and type of aminoacids, while a homologous or similar sequence may include conservativeor non-conservative amino acid changes without departing from theinventive concept. Thus, for example, a peptide having a singleconservative amino acid substitution may have higher similarity to theparent peptide than another peptide where that same single amino acid issubstituted with a non-conservative amino acid.

In many embodiments, specific amino acid positions and identities in asequence may be more important than other positions. In someembodiments, the importance of a position or amino acid may be analyzedby alanine-scanning or by multiple sequence alignment. For example, FIG.41 shows a multiple sequence alignment of the 19 amino acid peptidesequence of MMP13 with other similar sequences in MMP proteins. Thisalignment shows that the glycine at position 3 and the proline atposition 5 are both highly conserved within this family. Thus,maintenance of the position and identity of these two amino acids may bemore important than at other positions. Moreover, the N-terminus of thisembodiment of the inhibitory peptide may be more highly conserved thanthe C-terminus.

In many embodiments, the inventive compounds may be linear. In otherembodiments, the inventive compounds may be circular or cyclized bynatural or synthetic means. For example, disulfide bonds betweencysteine residues may cyclize a peptide sequence. Bifunctional reagentscan be used to provide a linkage between two or more amino acids of apeptide. Other methods for cyclization of peptides, such as thosedescribed by Anwer et. al. (Int. J Pep. Protein Res. 36:392-399, 1990)and Rivera-Baeza et al. (Neuropeptides 30:327-333, 1996) are also knownin the art.

The compounds of the invention may be modified with non-peptide moietiesthat provide a stabilized structure or lessened biodegradation. Peptidemimetic analogs can be prepared based on the compound of the presentinvention by replacing one or more amino acid residues of the protein ofinterest by non-peptide moieties. Preferably, the non-peptide moietiespermit the peptide to retain its natural conformation, or stabilize apreferred, e.g., bioactive conformation. One example of methods forpreparation of non-peptide mimetic analogs from peptides is described inNachman et al., Regul. Pept. 57:359-370 (1995). It is important that anymodification does not significantly reduce binding affinity of theinventive compound with its target binding substrate. In someembodiments, it may be useful to modify an inventive compound to achievegreater thermodynamic or degradative stability even though the bindingaffinity may be slightly compromised. The term “peptide” as used hereincan embrace non-peptide analogs, mimetics and modified peptides.

Peptidomimetics derivatives could be designed based on the two and threedimensions modeling of effective blocking peptides. A blocking peptidemay be used to describe an inventive compound with competes for bindingto MMP or a MMP substrate, resulting in disruption of the MMP-substrateinteraction. For example, MMP13-19 (amino acids 93-111 of SEQ ID NO: 1)has an alpha helical structure. Peptidomimetic derivatives of indanes,terphenyl, oligophenyls, chalcones, trans-fused polycyclic ethers couldbe used to design peptidomimetics with alpha helix backbone similar toMMP13-19. For beta-sheet peptides the following methods could be used;use of ferrocene amino acid conjugates where either peptide monomers(Henrick et al., Tetrahedron Lett. 37:5289-5292, 1996, which isincorporated herein by reference) or dimers (Moriuchi et al., J. Am.Chem. Soc. 123:68-75, 2001, which is incorporated herein by reference),the attachment of the peptides to the cyclopentadienyl core to generateeither ‘parallel’ or ‘anti-parallel’ strands (Barisic et al., Chem.Commun. 17:2004-2005, 2004, which is incorporated herein by reference)and using both covalent and non-covalent coordination methods tomaintain the β-sheet conformation beyond two residues.

The compounds of the present invention may be modified in order toimprove their efficacy. Such modification of the compounds may be usedto decrease toxicity, increase bioavailability, increase bindingaffinity, or modify biodistribution. A strategy for improving drugviability is the utilization of water-soluble polymers. Variouswater-soluble polymers have been shown to modify biodistribution,improve the mode of cellular uptake, change the permeability throughphysiological barriers, and modify the rate of clearance from the body(Greenwald et al., Crit Rev Therap Drug Carrier Syst. 2000; 17:101-161;Kopecek et al., J Controlled Release, 74:147-158, 2001). To achieveeither a targeting or sustained-release effect, water-soluble polymershave been synthesized that contain drug moieties as terminal groups, aspart of the backbone, or as pendent groups on the polymer chain.

For example, polyethylene glycol (PEG), has been widely used as a drugcarrier, given its high degree of biocompatibility and ease ofmodification (Harris et al., Clin Pharmacokinet. 2001; 40(7):539-51).Attachment to various drugs, proteins, and liposomes has been shown toimprove residence time and decrease toxicity (Greenwald et al., Crit RevTherap Drug Carrier Syst. 2000; 17:101-161; Zalipsky et al., BioconjugChem. 1997; 8:111-118). PEG can be coupled to active agents through thehydroxyl groups at the ends of the chain and via other chemical methods;however, PEG itself is limited to at most two active agents permolecule. In a different approach, copolymers of PEG and amino acidswere explored as novel biomaterials which would retain thebiocompatibility properties of PEG, but which would have the addedadvantage of numerous attachment points per molecule (providing greaterdrug loading), and which could be synthetically designed to suit avariety of applications (Nathan et al., Macromolecules. 1992;25:4476-4484; Nathan et al., Bioconj. Chem. 1993; 4:54-62).

The compounds encompassed by the present invention may also be attachedto magnetic beads or particles (preferably nano-particles) to controldistribution of the compound. Such compounds can specifically betargeted using a magnetic field, which naturally increases theeffectiveness of the compounds. Methods of attaching peptides tomagnetic beads are known in the art and are disclosed, for example inU.S. Pat. No. 5,858,534.

The compounds encompassed by the present invention may be produced byconventional automated peptide synthesis methods or by recombinantexpression. General principles for designing and making proteins arewell known to those of skill in the art.

The peptides encompassed by the present invention can be made insolution or on a solid support in accordance with conventionalFMOC-based techniques. The peptides can be prepared from a variety ofsynthetic or enzymatic schemes, which are well known in the art. Whereshort peptides are desired, such peptides are prepared using automatedpeptide synthesis in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and are used in accordance with known protocols. See, forexample, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed.,Pierce Chemical Co., (1984); Tam et al., J. Am. Chem. Soc., 105:6442,(1983); Merrifield, Science, 232:341-347, (1986); and Barany andMerrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, NewYork, 1-284, (1979); Fields, (1997) Solid-Phase Peptide Synthesis.Academic Press, San Diego); Andersson et al., Large-scale synthesis ofpeptides. Biopolymers (Pept. Sci.), 55, 227-250 (2000); Burgess et al.,J. Pept. Res., 57, 68-76, (2001); Peptides for the New Millennium,Fields, J. P. Tam & G. Barany (Eds.), Kluwer Academic Publisher,Dordrecht. Numerous other documents teaching solid phase synthesis ofpeptides are known to those of skill in the art and may be used tosynthesis epitope arrays from any allergen.

For example, the peptides are synthesized by solid-phase technologyemploying a peptide synthesizer, such as a Model 433A from AppliedBiosystems Inc. This instrument combines the FMOC chemistry with theHBTU activation to perform solid-phase peptide synthesis. Synthesisstarts with the C-terminal amino acid. Amino acids are then added one ata time till the N-terminus is reached. In some embodiments, non-naturalamino acids may be incorporated into a synthetically synthesizedpeptide. Three steps are repeated each time an amino acid is added.Initially, there is deprotection of the N-terminal amino acid of thepeptide bound to the resin. The second step involves activation andaddition of the next amino acid and the third step involves deprotectionof the new N-terminal amino acid. In between each step there are washingsteps. This type of synthesizer is capable of monitoring thedeprotection and coupling steps.

At the end of the synthesis the protected peptide and the resin arecollected, the peptide is then cleaved from the resin and the side-chainprotection groups are removed from the peptide. Both the cleavage anddeprotection reactions are typically carried out in the presence of 90%TPA, 5% thioanisole and 2.5% ethanedithiol. After the peptide isseparated from the resin, e.g., by filtration through glass wool, thepeptide is precipitated in the presence of MTBE (methyl t-butyl ether).Diethyl ether is used in the case of very hydrophobic peptides. Thepeptide is then washed a plurality of times with MTBE in order to removethe protection groups and to neutralize any leftover acidity. The purityof the peptide is further monitored by mass spectrometry and in somecase by amino acid analysis and sequencing.

The peptides also may be modified, and such modifications may be carriedout on the synthesizer with very minor interventions. An amide could beadded at the C-terminus of the peptide. An acetyl group could be addedto the N-terminus. Biotin, stearate and other modifications could alsobe added to the N-terminus.

The purity of any given peptide, generated through automated peptidesynthesis or through recombinant methods, is typically determined usingreverse phase HPLC analysis. Chemical authenticity of each peptide isestablished by any method well known to those of skill in the art. Incertain embodiments, the authenticity is established by massspectrometry. Additionally, the peptides also are quantified using aminoacid analysis in which microwave hydrolyses are conducted. In oneaspect, such analyses use a microwave oven such as the CEM Corporation'sMDS 2000 microwave oven. The peptide (approximately 2 μg protein) iscontacted with e.g., 6 N HCl (Pierce Constant Boiling e.g., about 4 ml)with approximately 0.5% (volume to volume) phenol (Mallinckrodt). Priorto the hydrolysis, the samples are alternately evacuated and flushedwith N². The protein hydrolysis is conducted using a two-stage process.During the first stage, the peptides are subjected to a reactiontemperature of about 100° C. and held that temperature for 1 minute.Immediately after this step, the temperature is increased to 150° C. andheld at that temperature for about 25 minutes. After cooling, thesamples are dried and amino acid from the hydrolysed peptides samplesare derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate toyield stable areas that fluoresce at 395 nm (Waters AccQ Tag ChemistryPackage). In certain aspects, the samples are analyzed by reverse phaseHPLC and quantification is achieved using an enhanced integrator.

In certain embodiments, the peptides of the present invention are madeusing FMOC solid-phase synthetic methods such as those described above.However, it is also contemplated that those skilled in the art mayemploy recombinant techniques for the expression of the proteins whereina nucleotide sequence which encodes a peptide of the invention isinserted into an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression as described herein below. Recombinant methods are especiallypreferred for producing longer polypeptides that comprise peptidesequences of the invention. Recombinant techniques are well known in theart. For example, U.S. Pat. No. 7,659,375 discloses several systems,including prokaryotic, yeast, mammalian and insect cell, for productionof recombinant peptides. As such, in an embodiment, nucleic acidsequences encoding the peptides or polypeptides of the presentinvention, and vectors containing those nucleic acid sequences are alsocontemplated.

In another embodiment, the present invention provides methods forinhibiting the ability of MMP13 to activate TGFβ by contacting the LTBP1with an inhibiting amount of a compound according to the presentinvention. Here, the compound of the present invention competes withMMP13 for interaction with LTBP1 thereby acting as a competitiveinhibitor of MMP13. By preventing MMP13 from binding LTBP1, the compoundof the present invention prevents cleavage of TGFβ LLC, and thereby,inhibits the activation of TGFβ. In that way, the methods of the presentinvention prevent activation of TGFβ without affecting the catalyticdomain of MMP13, thereby, avoiding problems associated with theinhibition of MMP13 by directly affecting its catalytic domain.

In anther embodiment, the present invention provides methods fortreating a mammal suffering from osteoarthritis or cartilagedegeneration by administering to the mammal a compound of the inventionin an amount sufficient to alleviate the effects of osteoarthritis orcartilage degeneration. Preferably, the compound is administereddirectly to the cartilage of the mammal, for example, by injection.

Specific amounts and route of administration may vary, and will bedetermined in the clinical trial of these agents. However, it iscontemplated that those skilled in the art may administer the compoundsof the present invention directly, such as by direct intra-jointinjection, to effect contact of the TGFβ LLC with the compounds toprevent activation of TGFβ. In a preferred embodiment, the compound ofthe present invention are administered so to achieve a concentration ofabout 10-250 nM, preferably 150-250 nM, of that compound in the synovialfluid.

In some embodiments, the inventive compound may be administered by atransdermal patch or topical lotion, balm, cream, etc. In otherembodiments, the inventive compound may be delivered systemicallythrough oral, nasal, or intravenous delivery.

Pharmaceutical compositions for administration according to the presentinvention can comprise the compound of the present invention alone or incombination with other therapeutic agents or active ingredients.Regardless of whether the active component of the pharmaceuticalcomposition is a compound alone or in combination with another activeagent, each of these preparations is in some aspects provided in apharmaceutically acceptable form optionally combined with apharmaceutically acceptable carrier. Those compositions are administeredby any methods that achieve their intended purposes. Individualizedamounts and regimens for the administration of the compositions for thetreatment of the given disorder are determined readily by those withordinary skill in the art using assays that are used for the diagnosisof the disorder and determining the level of effect a given therapeuticintervention produces.

Pharmaceutical compositions are contemplated wherein a compound of thepresent invention and one or more therapeutically active agents areformulated. Formulations of compounds of the present invention areprepared for storage by mixing said compound having the desired degreeof purity with optional pharmaceutically acceptable carriers, excipientsor stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed., 1980, incorporated entirely by reference), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,acetate, and other organic acids; antioxidants including ascorbic acidand methionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners andother flavoring agents; fillers such as microcrystalline cellulose,lactose, corn and other starches; binding agents; additives; coloringagents; salt-forming counter-ions such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG). In a preferred embodiment, thepharmaceutical composition that comprises the compound of the presentinvention may be in a water-soluble form, such as being present aspharmaceutically acceptable salts, which is meant to include both acidand base addition salts. “Pharmaceutically acceptable acid additionsalt” refers to those salts that retain the biological effectiveness ofthe free bases and that are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. The formulations to beused for in vivo administration are preferably sterile. This is readilyaccomplished by filtration through sterile filtration membranes or othermethods.

It is understood that the suitable dose of a composition according tothe present invention will depend upon the age, health and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired. However, the dosage is tailored tothe individual subject, as is understood and determinable by one ofskill in the art, without undue experimentation. This typically involvesadjustment of a standard dose, e.g., reduction of the dose if thepatient has a low body weight.

The total dose of therapeutic agent may be administered in multipledoses or in a single dose. In certain embodiments, the compositions areadministered alone, in other embodiments the compositions areadministered in conjunction with other therapeutics directed to thedisease or directed to other symptoms thereof.

In some aspects, the pharmaceutical compositions of the invention areformulated into suitable pharmaceutical compositions, i.e., in a formappropriate for applications in the therapeutic intervention of a givendisease. Methods of formulating proteins and peptides for therapeuticadministration also are known to those of skill in the art.Administration of those compositions according to the present inventionwill be via any common route so long as the target tissue is availablevia that route. Preferably, those compositions are formulated as aninjectable. Appropriate routes of administration for the presentinvention may include oral, subcutaneous, intravenous, transdermal,intradermal, intramuscular, intramammary, intraperitoneal, intrathecal,intraocular, retrobulbar, intrapulmonary (e.g., term release), aerosol,sublingual, nasal, anal, vaginal, or transdermal delivery, or bysurgical implantation at a particular site is also used, particularlywhen oral administration is problematic. The treatment may consist of asingle dose or a plurality of doses over a period of time.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all eases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Insome aspects, the carrier is a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity is maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms isbrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions is brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the compoundsof the present invention in the required amount in the appropriatesolvent with several of the other ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, themethods of preparation are vacuum-drying and freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution is suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. Those ofordinary skill in the art will readily optimize effective dosages andadministration regimens as determined by good medical practice and theclinical condition of the individual patient.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe compounds and the routes of administration. The optimalpharmaceutical formulation will be determined by one of skill in the artdepending on the route of administration and the desired dosage. Suchformulations may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the administered agents.Depending on the route of administration, a suitable dose is calculatedaccording to body weight, body surface areas or organ size. Theavailability of animal models is particularly useful in facilitating adetermination of appropriate dosages of a given therapeutic. Furtherrefinement of the calculations necessary to determine the appropriatetreatment dose is routinely made by those of ordinary skill in the artwithout undue experimentation, especially in light of the dosageinformation and assays disclosed herein as well as the pharmacokineticdata observed in animals or human clinical trials.

Typically, appropriate dosages are ascertained through the use ofestablished assays for determining blood levels in conjunction withrelevant dose response data. The final dosage regimen will be determinedby the attending physician, considering factors which modify the actionof drugs, e.g., the drug's specific activity, severity of the damage andthe responsiveness of the patient, the age, condition, body weight, sexand diet of the patient, the severity of any infection, time ofadministration and other clinical factors. As studies are conducted,further information will emerge regarding appropriate dosage levels andduration of treatment for specific diseases and conditions. Thosestudies, however, are routine and within the level of skilled persons inthe art.

It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention are useful in fields of humanmedicine and veterinary medicine. Thus, the subject to be treated is amammal, such as a human or other mammalian animal. For veterinarypurposes, subjects include for example, farm animals including cows,sheep, pigs, horses and goats, companion animals such as dogs and cats,exotic and/or zoo animals, and laboratory animals including mice, rats,rabbits, guinea pigs and hamsters.

In another embodiment, the present invention also provides antibodiesfor binding the compounds of the present invention which have MMP13inhibitory activity. The antibodies can be generated using the compoundsof the present invention as antigens in various methods known in theart. For example, the methods of U.S. Pat. No. 7,049,410, which isincorporated herein by reference, to make monoclonal and polyclonalantibodies can be used to make the antibodies against the compounds ofthe present invention. For example, a peptide having the amino acidsequence ELGLPKEVKKISAAVHFED (amino acids 93-111 of SEQ ID NO: 1,variously referred to as MPP13-19, pxpt 001-1, the “inhibitory peptide,”or the “peptide”) can be used as an antigen to generate monoclonal orpolyclonal antibodies to MMP13. The antibodies are useful asdiagnostics, e.g. in detecting the specific peptides or polypeptides ofthe present invention or MMP13, or determining the presence or absenceof the peptides or polypeptides in the body tissues. One skilled in theart would be able to utilize the antibodies in accordance with knowndiagnostic methods. In an embodiment, the antibodies may be labeled foreasy detection. The labels can be, but are not limited to biotin,fluorescent molecules, radioactive molecules, chromogenic substrates,chemi-luminescence, and enzymes.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds and methods of thepresent invention. The following examples are given to illustrate thepresent invention. It should be understood that the invention is not tobe limited to the specific conditions or details described in thoseexamples.

EXAMPLES Example 1 Materials and Methods

Cartilage extract preparation, immunoprecipitation and immunoblotanalysis—Tibia from normal Sprague-Dawley 14 day old newborn rat pups orday 19 chick embryos were dissected free of tissue and the tibial growthplates isolated by microdissection. The cartilage was minced andextracted overnight in 0.5%3-([3-chlomadipropyl]dimethylammonio)-1-propane-sulfonate (CHAPS) buffer[10 mM Tris, 100 mM NaCl, 2 mM EDTA, pH 7.6] (Sigma, St. Louis, Mo.,USA). Avian tissue was immunoprecipitated with rabbit anti human LTBP-1(a kind gift of Dr. Kohei Miyazono), rabbit anti avian MMP-13 (D'Angelo,et al., 2000) or rabbit pre-immune serum (Pierce Biochemicals, Rockford,Ill., USA). Rat tissue was immunoprecipitated with rabbit anti humanLTBP-1 or rabbit anti human TGFβ2 polyclonal antibodies (Santa CruzBiotechnology, Inc., Santa Cruz, Calif., USA). Concurrently, TGFβantibody was biotinylated using the EZ-Link Sulfo-NHS-LC-BiotinylationKit according to protocol (Pierce Biochemicals, Rockford, Ill., USA).Conditioned media from day 5 chondrocyte alginate cultures was incubatedwith biotin-labeled. TGFβ antibody for 30 minutes at room temperatureand passed over a μMACS strep-avidin micro-bead column (Miltenyi Biotec,Inc, Auburn, Calif., USA). All immunoprecipitates were separated on4-20% or 8-16% Tris-glycine, SDS-polyacrylamide gradient gels,(Invitrogen Life Technologies, Carlsbad, Calif., USA). Proteins weretransferred to Protran nitrocellulose (Schleicher and Schull, Keene,N.H., USA) in a Bio-Rad Mini-blot transfer apparatus (Bio-RadLaboratories, Inc., Hercules, Calif., USA), blocked for 2 hours at roomtemperature with 3% (wt/vol) nonfat milk in tris-buffered saline(TBS/Tween; 10 mM Trizma base (pH 8.0) and 150 mM sodium chloride and0.05% Tween-20), and incubated in TBS/Tween containing 1% nonfat milkand primary antibody raised against TGFβ2 (Santa Cruz Biotechnology,Inc: Santa Cruz, Calif., USA) or primary antibody raised against avianMMP-13 (D'Angelo, et al., J. Cell. Biochem. 77:678-693, 2000, which isincorporated herein by reference). Immunoblots were then exposed tohorseradish peroxidase-conjugated secondary antibody in TBS/Tween (SantaCruz Biotechnology, Inc., Santa Cruz, Calif., USA) and bands ofimmunoreactivity were visualized with the Western Blotting LuminolReagent (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) andexposed to Kodak X-Omat Blue XB-1 film (Kodak, Rochester, N.Y., USA). CMand CAM samples were concentrated 5-10 fold by centrifugation inCentricon concentrators (molecular weight (MW) cut-off=10 kDa), (PierceBiochemicals, Rockford, Ill., USA). Total protein was determined by themodified Lowry method (Pierce Biochemicals, Rockford, Ill., USA). 8-16%Tris-glycine, SDS-polyacrylamide gradient gels (Invitrogen LifeTechnologies, Carlsbad, Calif., USA) were loaded with 20 g total proteinper lane in Laemelli's reducing sample buffer and 8M urea. Immunoblotanalysis was conducted with primary antibody raised against aviancollagen type X (Pacifici et al., Exp. Cell Res. 192:266-270, 1991,which is incorporated herein by reference). The main band of collagentype X protein was scanned and analyzed with the Phase III Image Proanalyzer software (Phase III, Malvern, Pa., USA).

Serum-free Alginate Cultures—Chondrocytes were reared in serum-freealginate culture as previously described (D'Angelo et al., J. BoneMiner. Res. 16:2339-2347, 2001, which is incorporated herein byreference) Briefly, hypertrophic chondrocytes were isolated from day 17avian embryonic cephalic sternal core (following 5 days in culture theyare designated late hypertrophic (LH)) and cephalic sternal periphery(following 5 days in culture they are designated early hypertrophic(EH)) (D'Angelo et al., J. Bone Miner. Res. 12:1368-1377, 1997; andD'Angelo et al., 2001, which are incorporated herein by reference). Thetissue was enzymatically digested in 0.25% trypsin and 0.1% crudecollagenase and plated at final density of 5×106 cells/ml in 1.2%Keltone LVCR alginate (Kelco Clark, N.J., USA) or plated at 2×106cells/35 mm well for high-density monolayer cultures. CompleteSerum-free Media (DMEM-high glucose, 1 mM cysteine, 1 mM sodiumpyruvate, 50 μg/ml penicillin/streptomycin, and 2 mM L-glutamine)(Invitrogen Corporation) was added to the alginate cultures. Media waschanged and collected every 48 hrs with 30 μg/ml ascorbate added on day2 of culture. Conditioned media was pooled from alginate cultures on day5, the cells isolated by incubation with 55 mM sodium citrate and thesupernatant designated as cell-associated matrix. Pelleted cells wereextracted with 0.5% CHAPS detergent buffer [10 mM Tris, 100 mM NaCl, 2mM EDTA, pH 7.6) and designated cell layer fraction (SigmaBiochemicals). Conditioned media and cell-associated matrix samples wereconcentrated four-fold in Centricon-10 filters (MW cut-off 10,000Daltons) as per manufacturer's instructions (Fisher Scientific).

Reverse-transcription Polymerase Chain Reaction—Cells were isolated fromalginate culture following incubation in 0.5M EDTA, pH 8.0, then 5×106cells resuspended per ml of Trizol reagent (Invitrogen Corporation) andtotal RNA isolated as per manufacturer's instructions.Reverse-transcribed cDNA was prepared with Superscript enzyme accordingto manufacturer's protocol (Invitrogen Corporation) and subjected toamplification using Ready-to-go PCR beads per manufacturer'sinstructions (GE Healthcare). The following primers were designed fromthe NCBI database: avian 18srRNA Forward 5′-TTA ACG AGG ATC CAT TGGAG-3′ (SEQ ID NO: 21) Reverse 5′-AGC CTG CTT TGA ACA CTC TA-3′ (SEQ IDNO: 22); avian collagen type X Forward 5′-AGA GGA GTA CTC CTG AAA GT-3′(SEQ ID NO: 23) Reverse 5′-ACT GCT GAA CAT AAG CTC CT-3′ (SEQ ID NO:24); human LTBP-1short Forward 5′-CCG CAT CAA GGT GGT CTT TA 3′ (SEQ IDNO: 25) Reverse 5′-CAT ACA CTC ACC ATT AGG GC-3′ (SEQ ID NO: 26); humanLTBP-1 long Forward 5′-TGT GGA GGG CAG TGC TGC-3′ (SEQ ID NO: 27)Reverse 5′-TAA AGA CCA CCT TGA TGC GG-3′ (SEQ ID NO: 28); avian MMP-13Forward 5′-TAC TGC TGA TAT CAT GAT CTC-3′ (SEQ ID NO: 29) Reverse 5′-TCTAGA ATC ATC TGA CCA AGT-3′ (SEQ ID NO: 30); avian TGFβ2 Forward 5′-AATGAC AGC ATC AGG TAC GG-3′ (SEQ ID NO: 31) Reverse 5′-ATG GTC AGG ACT GAGGCA C-3′ (SEQ ID NO: 32); and avian caspase-3 Forward 5′-AGA TGT ATC AGATGC AAG ATC T-3′ (SEQ ID NO: 33) Reverse 5′-GAA GTC TGC TTC TAC AGGTAT-3′ (SEQ ID NO: 34). PCR products were separated on 2% agarose gels,densitometrically scanned (Gel-Pro Analyzer, Media Cybernetics, SilverSprings, Md., USA), and normalized to 18srRNA as an internal standard.Densitometric values for a minimum of n=3 were plotted using PrismGraphPad software version 3.03 (GraphPad Software, San Diego, Calif.,USA) with ANOVA and 95% C.I. Tukey's analysis.

Immunoblot Analysis—Immunoblot analysis was performed on the conditionedmedia (soluble fraction), cell-associated matrix fraction (territorialmatrix) and the CHAPS buffer extract (cell layer). 20 μg total proteinwas loaded per well, electrophoretically separated on precast 8-16%gradient Tris-glycine SDS polyacrylamide gels (Invitrogen Corporation),transferred to nitrocellulose, and incubated with rabbit polyclonalanti-TGFβ2 (Santa Cruz Biotechnology, Inc.), rabbit polyclonalanti-MMP-13 (D'Angelo et al., 2000), and rabbit polyclonal anti-LTBP-1(kind gift from K. Miyazono) followed by incubation with horse-radishperoxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).Reactive bands were visualized through chemiluminescence and exposedfilm densitometrically scanned (Gel-Pro Analyzer, Media Cybernetics,Silver Springs, Md., USA).

Immunocytochemistry—Chondrocytes were isolated and plated 2×106 cellsper 35 mm well onto 22×22 mm glass coverslips (VWR) and fed withComplete Media containing 10% NuSerum (Fisher Scientific). Cultures wereincubated up to 48 hours until confluent, in the presence of 40 nghyaluronidase per milliliter complete media. Cells were fixed inCytoFix/CytoPerm (BD Biosciences) per manufacturer's instructions andcell-free extracellular matrix samples were produced by lysingchondrocytes with cold phosphate buffered saline and 10% Triton X-100 asdescribed (Dallas et al., J. Cell Biol. 131:539-549, 1995, which isincorporated herein by reference). Cells and extracellular matrix wereincubated with primary antibodies (Santa Cruz Biotechnology): 100 mg/mlIgG TGFβ2 (V), 20 μg/ml IgG MMP-13 (E-20), and 20 μg/ml IgG LTBP-1(N-20) followed by secondary antibodies: 1 ng/ml goat anti-rabbit AlexaFluor 594 rhodamine in the case of TGFB and MMP-13 or 1 ng/ml donkeyanti-goat Alexa Fluor 488 fluoroscene in the case of LTBP-1 (Santa CruzBiotechnology). Nuclei were counterstained with 1 μg/ml Hoechst dye(Sigma-Aldrich), fluorescent images were captured with Nikon E600fluorescent microscope (Nikon Inc., Melville, N.Y., USA) at 100×objective, and z-stacks acquired with ImagePro 4.5 (Media Cybernetics,Silver Springs, Md., USA) then deconvoluted with AutoDeblurAutoVisualize version 9.2.1 software (AutoQuant Imaging Inc.,Watervliet, N.Y., USA).

Results

A novel TGFβ large latent complex produced by hypertrophic chondrocytescontains MMP-13—Early and late hypertrophic chondrocytes produceactivated TGFβ and another hypertrophy-specific marker, themetalloprotease, MMP-13, has a role in the activation of TGFβ byhypertrophic chondrocytes (D'Angelo et al., 2001). Immunoblot analysisof conditioned media from early and late hypertrophic chondrocytealginate cultures revealed that antibody to MMP-13 cross-reacted with a280-300 kDa, putative TGF-β2 LLC complex (data not shown). To elaboratethis observation, we prepared extracts from day 19 avian tibialhypertrophic chondrocytes, immunoprecipitated proteins with MMP-13 orLTBP-1 antibody and then subjected the immunoprecipitates to immunoblotanalysis with TGFβ2 (FIG. 2A). TGFβ-immunoreactive bands were detectedat approximately 290 kD, that is the putative large latent TGFβ complexproduced by hypertrophic chondrocytes, 60 kD representing the N-terminalβ-LAP fragment of TGFβ□ and 25 kD representing the homodimer ofactivated TGFβ□ whether immunoprecipitated with αLTBP-1 or αMMP-13antibody (FIG. 2A). Rat tibial growth plate cartilage extractsimmunoprecipitated with antibody to TGFβ2 contained MMP-13immunoreactive bands at approximately 130 kDa representing the solubleform of the TGF-β large latent complex and 68 kDa and 52 kDarepresenting the proenzyme and activated enzyme forms of MMP-13 (FIG.2B). Conditioned media from late hypertrophic chondrocyte alginatecultures were immunoprecipitated with biotin-labeled polyclonal antibodyto TGFβ and immunoblotted with αMMP13 revealing an approximate 52 kDaimmunoreactive band (FIG. 2C). These data indicate the production of aunique TGF-β LLC produced by mammalian and avian hypertrophicchondrocytes that includes MMP-13 in non-covalent association.

MMP-13 associates with hypertrophic chondrocyte produced TGF-β LLC—Totalmessenger RNA was examined in the alginate cultures to confirm that bothpopulations of cells were hypertrophic (FIG. 3A). Indeed, expression ofcollagen type X mRNA and MMP-13 mRNA, markers of hypertrophy; wasobserved at high levels in both early and late hypertrophic chondrocytes(EH and LH, respectively) even though they had not progressed toterminal differentiation as evidenced by low levels of mRNA expressionfor caspase-3, an apoptotic marker (FIG. 3A). Both populations expressedmessage for LTBP-1 and TGFβ, components of the TGF-β LLC (FIG. 3B). Ithas been shown by other laboratories that LTBP-1 can be alternativelyspliced to create a long form that maintains a complete N-terminus, or ashort from that possesses a truncated N-terminus thought to be moreeasily removed from the extracellular matrix. We designed primers todifferentiate between both forms of LTBP-1 and demonstrated thathypertrophic chondrocytes produce both the long and short forms ofLTBP-1 (FIG. 3B) with late hypertrophic chondrocytes producing five-foldmore of the short form of LTBP-1 than early hypertrophic chondrocytes.

We conducted immunoblot analysis on day 5 alginate chondrocyte culturesto ascertain what forms of the TGF-β LLC are produced and secreted.Protein components of the TGF-β LLC were identified either in thesoluble conditioned media fraction (soluble) or the incorporatedproteins of the extracellular matrix fraction (territorial matrix) orthe proteins associated with the CHAPS-extracted cell pellet (celllayer) (FIG. 4). Reduced immunoblot analysis utilizing antibodies toTGFβ2 (FIG. 4A), MMP-13 (FIG. 4B), and LTBP-1 (FIG. 4C) revealed thepresence of these proteins in all three fractions examined. Latehypertrophic chonchocytes, overall, produced more of the three proteinsthan did early hypertrophic chondrocytes. TGFβ antibody cross-reactedwith bands at approximately 240 kDa, the TGF-β LLC, 130-190 kDa, theputative soluble species of the TGF-β LLC, 100 kDa, the TGF-β smalllatent complex, 60 kDa, the putative β-LAP, and 25 kDa, the TGFβbioactive homodimer (FIG. 4A). The majority of detectable protein waspresent in the soluble and cell layer fractions indicating hypertrophicchondrocyte production and secretion of the TGF-β large and small latentcomplexes. In addition, late hypertrophic chondrocyte samples producedmore TGFβ immunoreactive protein per total protein than did earlyhypertrophic chondrocytes (FIG. 4A, EH versus LH).

MMP-13 immunoblot analysis revealed an approximate 64 kDa band, theMMP-13 proenzyme and a 52 kDa band representing the active MMP-13 enzyme(FIG. 4B). In addition, a less intense immunoreactive band was visibleat 130-190 kDa, representing the putative soluble species of TGF-β LLCand a detectable band at approximately 240 kDa representing the TGF-βLLC (FIG. 4B). LTBP-1 immunoblot revealed an 80 kDa band of LTBP-1 and aless intensely stained band of 240 kDa, the TGF-β LLC (FIG. 4C). Thepresence of a 130-190 kDa band in the soluble layer indicates productionof the soluble species of the TGF-β LLC. In the LTBP-1 immunoblots,bands were detectable in the territorial matrix, as well as the secretedand cell layer fractions, indicating incorporation of LTBP-1 into theextracellular matrix produced by hypertrophic chondrocytes. Takentogether, the immunoblot data suggest association of MMP-13 with theTGF-β LLC.

Extracellular immunolocalization of the hypertrophicchondrocyte-produced TGF-β LLC—Preparation of cell-free matrices fromhigh-density plating of late hypertrophic chondrocytes was confirmed bycollagen type X staining (FIG. 5). Cell-free extracellular matricesexhibited strong collagen type X labeling (FIG. 5A) compared tosecondary antibody control (FIG. 5C) and cell cytoplasm stainedintensely for collagen type X (FIG. 5B, arrow). Cytoplasmic andextracellular staining of collagen type X was confirmed bythree-dimensional z-stack construction: two-dimensional slices weretaken at stepped focal planes within the cell and then compiled to yieldthree-dimensional representations that can be rotated around an axis(FIG. 5D).

TGFβ2, MMP-13, and LTBP-1 were all present in high-density monolayers oflate hypertrophic chondrocytes. All three proteins were observed withinthe extracellular matrix (FIG. 6A-C, arrows) and cytoplasm of thehypertrophic chondrocytes. Rotating three-dimensional z-stacks confirmedimmunolocalization of all three proteins in the cytoplasm of the latehypertrophic chondrocytes, and extracellular matrix staining wasdetectable in the images after background subtraction. Markedextracellular matrix staining of LTBP-1 was evident, whereas, MMP-13staining was more punctate and TGFβ staining more diffuse in theextracellular matrix (FIG. 6D-F).

Co-localization of MMP-13 and LTBP-1 in the hypertrophicchondrocyte-produced TGF-β LLC—Previous bioinformatics work from ourlaboratory indicated a plausible interaction of the hernopexin domainsof MMP-13 with the calcium-EGF-like domains of LTBP-1 (Selim et al., JBone Miner. Res. 20:S131, 2005; and Mattioli et al., J. Bone Miner. Res.19:S216, 2004, which are incorporated herein by reference). To confirmthis hypothesized interaction, we utilized immunocytochemical stainingmethods to co-localize MMP-13 and LTBP-1. Composite image overlay ofMMP-13 and LTBP-1 staining confirmed co-localization of the two proteinsobserved as yellow-orange staining (FIG. 7C1-7C3). The overlay indicatesthat the proteins of interest are within close proximity and thatstaining is within the cytoplasm (FIG. 7C1, arrow) and within theextracellular matrix (FIG. 7C3, arrow). Rotated three-dimensionalz-stacks confirmed co-localization of MMP-13 and LTBP-1 as evidence bymost robust yellow to orange staining within the cytoplasm andextracellular matrix (FIG. 7D).

The data presented in this study support a mechanism for hypertrophicchondrocyte activation of the TGFβ LLC (FIG. 1). In this model, MMP-13produced by hypertrophic chondrocytes interacts with the LTBP-1 portionof the TGF-β LLC. Once non-covalently associated with the TGF-β, LLC,MMP-13 cleaves the LTBP-1 protease-sensitive hinge region and releasethe soluble 130-190 kDa TGFβ LLC. This soluble form of TGFβ LLC would bemore susceptible to proteolytic release of the TGFβ SLC and subsequentactivation of the TGFβ homodimeric form. Even though the antibodiesutilized in our immunocytochemical studies do not differentiate betweenthe 130-190 kDa soluble species of the TGF-β LLC and the full lengthTGF-β LLC, we expect that MMP-13 cleavage of the protease-sensitivehinge region of LTBP-1 could occur both in the extracellular matrix(most likely site) and within the cell cytoplasm. Because our modelincludes a non-covalent interaction between LTBP-1 and MMP-13, thedriving force for the formation of the unique TGFβ LLC would beconcentrations of the molecules MMP13 and TGF-β LLC within closeproximity to one another.

Example 2 Materials and Methods

Avian chondrocyte isolation and serum-free cell culture—Sterna wereremoved from day 17 chick embryos using microsurgical techniques aspreviously described (D'Angelo et al., J. Bone Miner. Res. 12:1368-1377,1997). Cells were released by digestion of the extracellular matrix in0.25% trypsin and 0.1% crude collagenase mixture (Sigma, St. Louis, Mo.,USA) in Hanks' buffered saline solution for 3-4 hours at 37° C.Digestion was halted by suspension in high glucose Dulbecco's modifiedEagle's medium (DMEM) containing 10% fetal bovine serum (FBS)(Invitrogen Life Technologies, Carlsbad, Calif., USA). The cellsuspensions were then filtered through a 0.2 μm filter, counted, seededonto Falcon 6-well, tissue culture treated plates and covered with 2 mlof high glucose DMEM containing 10% NuSerum and 50 μg/ml ofpenicillin/streptomycin, 2 mM of L-glutamine (Invitrogen LifeTechnologies, Carlsbad, Calif., USA). After 48 hours the cells wererinsed and processed for binding studies as described below.

Peptide binding assay—Twenty-five micrograms of total equal protein fromcartilage CHAPS extracts or five nanograms recombinant protein of thecalcium-like EGF binding domain of LTBP1 (a kind gift of Sarah Dallas)or hypertrophic cartilage from day 17 avian embryo sterna and tibialgrowth plate cartilage from day 14 newborn rat pups were extracted with0.5% CHAPS buffer and eluates immunoprecipitated with polyclonalantibody to LTBP1 and then samples were plated onto poly-L-lysine coated96 well plates. After overnight drying, the protein-coated wells wereincubated with 100 nM fluorescent-labeled peptide in Tris bufferedsaline [150 mM NaCl, 10 mM Tris, pH 8.0] and 20% horse serum for 4 hoursat 16° C. After incubation, the wells are washed twice with bindingbuffer, the protein layer solubilized with 5N NaOH and fluorescencebound measured at 495 nm excitation and 520 nm emission on theLabSystems Fluoroskan Ascent CF plate reader. Statistical analyses andgraphing were performed with Prism GraphPad software (ANOVA withTurkey's Analysis). A scrambled peptide of the same amino acidcomposition was prepared as a negative control. Competition assays wereconducted in the presence of 10-fold more (1 μM) naked peptide.

Bioinformatics analysis—Amino acid sequences of proteins analyzed in themanuscript were obtained from Swiss-Prot data base(http://www.ebi.ac.uk/swissprot/). Pfam data base(http://www.sanger.ac.uk/Software/Pfam/) was used for protein domainanalysis. Pfam-A is based on hidden Markox model (HMM) searches, asprovided by the HMMER2 package (http://hmmer.janelia.org/). In HMMER2,like BLAST, E-values (expectation values) are calculated. The E-value isthe number of hits that would be expected to have a score equal orbetter than this by chance alone. A good E-value is much smaller than 1because 1 is what is expected that sequences are similar by chance. Inprinciple, the significance of a match is predicated on a low E-value.3D models were generated using I-TASSER database(http://zhang.bioinformatics.ku.edu/I-TASSER/). Protein docking modelswere generated using Vakser lab database(http://www.vakser.bioinformatics.ku.edu/resources/gramm/grammx/).Protein-protein interface prediction data was generated using PIP-Preddatabase (http://bioinformatics.leeds.ac.uk/ppi_pred/index.html). Imageswere generated using Jmol software (http://jmol.sourceforge.net/).

Results

Model of the novel TGFβ large latent complex produced by hypertrophicchondrocytes—To examine the nature of interaction between MMP13 andLTBP1, we analyzed their structural domains. MMP13 has a peptidase-likedomain (P) and three hemopexin-like domains (H) (Table 1 and FIG. 8A).

TABLE 1 Domain MMP13 (avian) Sequence ^(a)E-Value Peptidase (P)  19-1748.2e−104 Hemopexin (H) 197-239 6.9e−12 241-284 3.5e−10 289-336 2.5e−14LTBP1 short (human) Cysteine-rich (C) 687-728 4.4e−23 1358-1401 9.4e−20^(b)1535-1577  2.1e−20 EGF-like (E) 191-218 0.00024 403-430 3.7e−61626-1661 8.6e−5 Calcium-binding EGF-like 626-665 2.7e−9 (CE) 916-9562.7e−14 958-997 8.6e−14  999-1037 3.9e−10 1039-1078 2e−9 1080-1119 Ie−101121-1160 3.9e−10 1162-1201 6.3e−14 1203-1243 1.2e−11 1245-1285 4.6e−111249-1285 1.6e−8 1287-1328 0.00033 1425-1466 0.0003 1468-1507 0.000121663-1706 3.2e−9 ^(a)The expectation values (E-value) of the homology ofthe regions of these molecules was determined as described in theMaterials and Methods section. The lower the E-value the more likely thesequence is a specific match. ^(b)Known sequence for the cysteine-richarea Where the TGFβ small latent complex covalently binds to LTBP1

The hemopexin-like domain is important for substrate specificity. Italso facilitates binding to a variety of molecules and proteins, forexample the hemopexin repeats of some matrixins bind tissue inhibitor ofmetalloproteases (TIMPs). LBTP1 is a larger molecule that consists ofseveral domains: EGF-like domain (E), calcium-binding EGF-like domain(CE) and cysteine rich (the TGFβ small latent complex binding) domain(C) (Table 1 and FIG. 8A). The role of the calcium-binding EGF-likedomain varies, depending on the function of the parent molecule, but itappears to be primarily involved in inter-domain interactions betweensome proteins. In addition, LBTP1 has a linker region that is sensitiveto proteolytic cleavage.

The data suggests a model in which the hemopexin domain of MMP13interacts with calcium-binding EGF-like domain of LBTP1 short in anorientation that places the peptidase domain of MMP13 at very closeproximity to the linker region of LBTP1 short (FIG. 8B). This puts MMP13in the correct conformation to line up the highly conserved sequence(HEXGHXXGXXHS/T) the catalytic domain of MMP13 (FIG. 8B, P) with theprotease-sensitive hinge region of LTBP1 short (FIG. 8B, Linker region).This is the site thought to be the target for release of LTBP1 from theextracellular matrix. Furthermore, this orientation is supported bypresence of candidate amino acids in this region of LTBP1 short, Gly-Ilebonds at positions 807/808 and 819/820, that are known to interact withthe peptidase domain of MMP13.

As a result of these predictions, we embarked on a bioinformatics studyto determine the potential interaction between MMP13 and LTBP1. Thepotential interaction of MMP13 hemopexin domains with the EGF-likecalcium binding domains of LTBP1 was indicated by a protein databasefile generated by the protein docking program Vakser lab. Modeling witha protein-protein docking program resulted in a three-dimensional modelthat corroborates the MMP13 molecule interaction with LTBP1 (FIGS. 8Aand B) and the site of interaction is toward the N-terminus, not the Cterminal site of TGFβ linkage to LTBP1 (FIG. 8B). Binding motifsanalysis within the MMP13-LTBP1 complex demonstrated a high affinityinterfacing area within the catalytic domain and moderate affinityinterfacing area within hemopexin like domains (FIG. 8C). In order todemonstrate these predicted interactions, we designed three candidatepeptides from the hemopexin domain of MMP13 that could potentiallyinteract non-covalently with the CE region of LTBP1.

Peptides designed to MMPI3 hemopexin domain specifically bind LTBP1proteins—We conducted binding studies with avian cartilage tissueextracts from the resting and hypertrophic zones of the cartilage growthplate. Both tissues produce TGFβ and store it in the extracellularmatrix in the form of the LTBP1-containing TGFβ large latent complex.However, hypertrophic cartilage tissue produces more TGFβ, a largerpercentage of which is activated, than that stored in the restingcartilage. In addition, only hypertrophic chondrocytes produce MMP13,thus offering us a model to compare different chondrocyte-producedcartilage tissue and its subsequent binding to MMP13-derived peptides.

MMP13-19 peptide (amino acids 93-111 of SEQ ID NO: 1) bound to thehypertrophic cartilage tissue extract approximately nine-fold more thanscrambled peptide control, as compared to a five-fold binding of restingcartilage tissue (FIG. 9B and A, respectively). MMP13-10 peptide (aminoacids 17-26 of SEQ ID NO: 1) bound less than two-fold the scrambledpeptide whether it was the resting or hypertrophic cartilage tissue(FIG. 9C) and MMP13-6 peptide (amino acids 37-42 of SEQ ID NO: 1) didnot specifically bind either cartilage tissue (FIG. 9D). All binding wascompeted with 10-fold excess non-fluorescent peptide.

To determine binding of the peptides to LTBP1 in its nativeconformation, hypertrophic chondrocytes were plated in monolayer toproduce a native extracellular matrix. MMP13-19 and MMP13-10 peptidesbound to the extracellular matrix of whole cell primary chondrocytecultures eight-fold and four-fold, respectively, compared to scrambledpeptide (FIG. 10A). Again, MMP13-6 did not bind specifically (data notshown).

Since total cell extracts and whole cell cultures contain more proteinsthan just LTBP1, we conducted binding studies on cartilage tissuesamples immunoprecipitated with antibody to LTBP1. MMP13-19 peptidebound 10.5-fold more than scrambled control in the hypertrophiccartilage immunoprecipitates (FIG. 10B). Binding with rat tibial growthplate cartilage immunoprecipitated extracts was 3.9-fold higher thanscrambled control (FIG. 10B) demonstrating a global interaction and nota species-specific binding between LTBP1 and the MMP-13 derived peptide.

To determine whether binding is occurring at the calcium-bindingEGF-like domains of LTBP predicted by our bioinformatics model, weconducted binding studies with a recombinant protein designed from thisregion (CE-LTBP1). In these studies, MMP13-19 peptide bound 27-fold morerecombinant protein than the scrambled peptide, whereas MMP13-10 andMMP13-6 did not bind specifically (FIG. 10C).

Example 3

To measure the ability of MMP13-19 peptide to bind endogenous largelatent complex of TGFβ and interfere with the activation of this growthfactor, hypertrophic chondrocytes were cultured in alginate beads inserum-free medium for 24 hours with varying concentrations of MMP13-19peptide. Conditioned media was then subjected to an ELISA to measuretotal TGFβ produced and the percentage of endogenously activated TGFβ(FIG. 11). All three doses (10, 100, and 750 nM) of MMP13-19 peptideresulted in a statistically significant decrease in endogenouslyactivated TGFβ although the total amount of TGFβ produced was notaffected. These data indicate that the MMP13-19 peptide can be used asan inhibitor of TGFβ activation.

Example 4

Animals were treated with MMP13-19 peptide or BMP-7 protein once a weekfor two (d14) or three (d21) weeks following an injection ofmono-iodoacetate (MIA), a chemical agent that induces osteoarthritispathology measurable at four weeks post injection. The samples thatreceived saline are the positive disease control. 250 nM of MMP13-19peptide and 50 uM BMP-7 were injected laterally below the patellarligament. BMP-7 has been shown to be chondroprotective in a similarmodel of OA. But, it is also known that BMP-7 is bone-inducing. The dataare shown in Tables 2-3 below.

TABLE 2 Total Volume Bone Volume BV/TV Patellar cartilage d14 OA saline8.0451 0.1486 0.0185 OA MMP13-19 peptide 9.1804 0.1247 0.0136 OA BMP-76.6967 0.419 0.0626 Patellar cartilage d21 OA saline 6.0544 0.32040.0529 OA MMP13-19 peptide 7.7109 0.1912 0.0248 OA BMP-7 10.7748 0.20370.0192

TABLE 3 Total Volume Bone Volume BV/TV Total Joint Cartilage d14 OAsaline 24.9735 0.5067 0.0243 OA MMP13-19 peptide 22.2703 0.6334 0.0284OA BMP-7 24.4891 1.0643 0.0435 Total Joint Cartilage d21 OA saline24.5334 1.3883 0.0566 OA MMP13-19 peptide 24.367 0.6164 0.0253 OA BMP-723.3101 0.9607 0.0412 NOTE: A lower value for Bone Volume or TotalVolume or a lower BV/TV ratio indicates cartilage that is NOTmineralized.

The amount of bone volume present in the samples indicates the areas ofmineralization. Since cartilage is not normally mineralized, one wouldexpect a low bone volume in these samples. In OA, cartilage will beginto mineralize. Of the conditions tested in this preliminary study,MMP13-19 peptide was the most effective at maintaining a normal range ofcartilage with the lowest bone volume at each time point. This indicatesa chondroprotective function for the MMP13-19 peptide that is evenbetter than the known effects of BMP-7.

Example 5 Peptide Interaction with Collagen

Bioinformatics may be used to identify candidate fragments that caninteract with substrates. For example, a three-dimensional model of dogMMP13 was generated. The dog MMP13 3D structure was docked with the 3Dstructure of substrate (such as type II collagen). The 3D structure ofthe complex was generated and then analyzed to identify interfacingresidues in MMP13 and substrate (such as type II collagen). Peptidesdesigned based on the interfacing residues (derived from MMP13 or thesubstrate) could be used to modify the interaction between MMP13 and itssubstrates. Preferably, the fragment contains at least 10 amino acids,more preferably 10 to 40 amino acids,

Dog MMP13 amino acid sequence was obtained from UniProt database (dogsequences may be found at SEQ ID NOs:124-150). The chain was used togenerate three dimensional (3D) model of dog MMP13 (FIG. 13). The 3Dstructure is showing the typical known structure of MMPs withcollagenase domain toward the N-terminus. This is the first time tomodel dog MMP13. Collagen is a known substrate for differentcollagenases. The 3D structures of MMP13 and collagen triple helixcomplex (FIG. 14) was generated using protein docking servers. Thecomplex demonstrated sandwich-like structure where collagen is lyingwithin a groove within MMP13 (FIG. 15). The complex was visualized andinteracting residues were identified. Peptide-based compound wasdesigned and modeled (FIG. 16). To test the ability of the peptide tointerfere with MMP13-collagen interaction, we docked MMP13, collagen andpeptide together in one complex (FIG. 17). Complex modeling demonstratedthat MMP13 derived peptide is interrupting the MMP13-collagenaseinteraction by interfering with the sandwich orientation (FIGS. 16 and17). Sequences used for these figures, and other dog sequences are givenin SEQ ID NOs: 124

These data suggested that such peptide has a potential competitiveinhibitory effect on MMP13-collagen interaction. 124-151.

Example 6 Peptide Interaction with Aggrecan Molecule

Aggrecan is a known substrate for MMP13. Aggrecan is a major componentof cartilage matrix. The peptide was docked with aggrecan (FIG. 18).Complex energy was −0.6 and two hydrogen bonds are predicted in thecomplex.

Example 7 Peptide Interaction with Fibronectin

Fibronectin is another known substrate for MMP13. The peptide was dockedwith fibronectin III (FIG. 19). Complex energy was −6.9 and threehydrogen bonds are predicted (FIG. 19).

Example 8 MMP13 Cleavage of LTBP1

Since TGFβ activation can be altered by competitive inhibition of theendogenous MMP13, then MMP13 should be able to utilize theprotease-sensitive hinge region of LTBP1 as a substrate as indicated byour working model (FIG. 1). In order to test this, we utilized afluorescence labeled peptide of the published protease-sensitive hingeregion for LTBP1, REHGARS (Taipale, J; Miyazono, K; Heldin, C-H and J.Keski-Oja (1994) JCB 124, 171-181). Enzyme kinetic assays with acommercially available MMP13 catalytic domain (Enzo, Inc) were conductedwith our hinge region peptide substrate. Michaelis-Menten non-linear fitfor the MMP13 digest of the hinge substrate demonstrates a Km=1.628e-016(FIG. 21A). When this activity is compared with a peptide substrate ofscrambled sequence, the two lines have statistically significantdifferences in slopes (p<0.03419) (FIG. 21B). These data indicate thatMMP13 can utilize the protease-sensitive hinge region of LTBP1 as asubstrate. Thus, our modeled interaction of MMP13 with the TGFβ largelatent complex could be a method of release of TGFβ from extracellularmatrix stores.

Utilizing a standard curve with MMP13 and MMP9 catalytic domains (Enzo,Inc) we quantitated the amount of MMP13 and MMP9 activity in cartilageextracts (Table 4). Cartilage isolated from day 17 avian embryos contain0.166 units and 0.152 units MMP13 per 40 μg total tissue in earlyhypertrophic and late hypertrophic tissue, respectively. As expected,resting cartilage does not contain measurable quantities of MMP13activity. Inclusion of an inhibitor of MMP13 (Calbiochem) reduces theactivity in both cartilage populations by 24% and 30% respectively.

All of the cartilage extracts contain enzymatic activity that can digestthe hinge substrate (FIG. 22). MMP13 catalytic domain and resting, earlyand late hypertrophic cartilage all contain enzymatic activity that isstatistically significant when compared to the scrambled substrate (FIG.22A). The amount of activity in the cartilage extracts is significantlyhigher than the MMP13 catalytic domain alone (FIG. 22B) indicating thatagents other than MMP13 are responsible for these data. This is furthersupported by the statistically significant activity present in restingcartilage, a tissue that does not contain MMP13 activity (see Table 4).

TABLE 4 MMP13 Enzymatic Activity in Cartilage Extracts. *Units MMP13MMP13 Sample ID Activity Inhibitor MMP13/9 Inhibitor Restingchondrocytes 0 Early Hypertrophic 0.166 −23.68% −20.39% Late Hypertophic0.152 −29.50% −27.50% Table 4: CHAPS extracts of avian sterna cartilagewere prepared from day 17 embryos. After dialysis with PBS, 20 ug totalprotein was assayed with MMP13 substrate (Enzo Laboratories). An MMP13catalytic domain standard curve was prepared. Michaelis-Menten enzymekinetics activity was calculated and units of enzyme activityinterpolated from the MMP13 catalytic domain standard curve. Inhibitorsof MMP13 and MMP13/9 activity were included in the assay (Calbiochem).n > 3 separate extractions was tested. All statistics were calculatedwith Prism GraphPad software. *40 μg total protein

Example 9 Chronic OA Pathology in the Articular Cartilage of theTibial-Femoral Joint Space

Rats were injected below the patella with mono-iodoacetate to induce OApathology. One week following the initial insult, rats were injectedwith saline (Control OA) or 250 nM pxtx001-1 peptide (Peptide treatedSEQ ID 36 (FIG. 22A) every other week out to 12 weeks. Rats weresacrificed and joints collected, dissected free of tissue, fixed informalin, decalcified, paraffin-embedded, sectioned and stained withhematoxylin and eosin (H&E) or saffranin O (for total proteoglycancontent). Grading was assessed by the OARSI (Pritzker, K. P., Gay, S.,Jimenez, S. A., Ostergaard, K., Pelletier, J. P., Revell, P. A., Salter,D., and van den Berg, W. B. (2006) Osteoarthritis Cartilage 14, 13-29)scale for pathology. FIG. 23 shows histopathology results of the presentexperiment comparing normal (top), control (middle), and peptideinhibitor treated (bottom) stained with both safranin-O (right), andhematoxaylin+eosin (left). Histologically, the peptide treated jointshave a lower grading on the OA scale than the untreated joints asevidenced by proteoglycan content and abnormal chondrocyte morphology.

Example 10 Acute Treatment of Osteoarthritis Model with High and LowDose Peptide Inhibitor

We have utilized an experimental rat model of OA by injection ofmono-iodoacetate (MIA) through the infrapatellar ligament of 150 g,male, Wistar rats. This model is characterized by osteophyte formationat the joint edges, fibrillation and erosion of the cartilage andsclerosis of the subchondral bone within 30 days of the injection(Janusz, M. J., Hookfin, E. B., Heitmeyer, S. A. et al. (2001)Osteoarthritis Cartilage 9, 751-760, and Guingamp, C., Gegout-Pottie,P., Philippe, L., Terlain, B., Netter, P., and Gillet, P. (1997)Arthritis Rheum. 40, 1670-1679). We analyzed joint cartilage pathologyin the MIA-induced OA model following injection of candidate peptides.

MIA (3 mg in 50 ul) was injected into the capsule of the stifle throughthe infrapatellar ligament of the right knee (Janusz, M. J., Hookfin, E.B., Heitmeyer, S. A. et al. (2001) Osteoarthritis Cartilage 9, 751-760,and Guingamp, C., Gegout-Pottie, P., Philippe, L., Terlain, B., Netter,P., and Gillet, P. (1997) Arthritis Rheum. 40, 1670-1679). Contralateralknees were injected with saline to serve as control for the experiment.Disease parameters were clearly measurable within three to four weeksfollowing injection. Animals were injected weekly with various doses ofpeptide (SEQ ID 36 (FIG. 20A)), beginning with the concentration thatwas shown to be effective in in vitro assays, 250 nM. Saline and BMP-7(500 ng=50 uM) were injected for negative and positive controls,respectively. Joints were X-rayed to measure joint space changes as anindicator of the progression of OA (Messent, E. A., Ward, R. J., Tonkin,C. J., and Buckland-Wright, C. (2005) Osteoarthritis Cartilage 13,463-470).

All animals were sacrificed 1, 2 and 3 weeks post injection of MIA.Isolated joints were analyzed by Micro CT to measure cortical bone,trabecular bone and cartilage of the patella, femur and tibia, theproduction of chondrophytes and tissue mineralization in response totreatment. Total mineralization in the patella, femur and tibialcartilages, as well as subchondral bone, was calculated with Scanco μCTsoftware. Micro-CT was conducted with a Scanco uCT 35 (Scanco Medical,Bassersdorf, Switzerland) system. Scans of 15 μm voxel size, 55 KVp,0.36 degrees rotation step (180 degrees angular range) and a 600 msexposure per view will be produced from joints immersed in phosphatebuffered saline.

Whole patella for total, cortical and trabecular bone, 3 mm of bothdistal femur and proximal tibia for cancellous bone, the individuallydefined volume between patella and femur and fixed volume of jointbetween femur and tibia were evaluated. The Scanco μCT software (HP,DECwindows Motif 1.6) was used for 3D reconstruction and viewing ofimages. Volumes were segmented using a global threshold of 0.4 g/c forbone and 0.25 g/c for soft tissue. Cortical bone was evaluated fortissue mineral density (TMD) and thickness of the cortex. Bone volumefraction (BV/TV), surface to volume ratio (BS/BV), thickness (Tb.Th),number (Tb.N) and separation (Tb.Sp) was calculated for the trabecularbone. Cartilage was analyzed for total volume (TV), mineral to totalvolume ratio (BV/TV) and apparent mineral density.

TABLE 5 Acute Treatment Week 4 Micro CT (n > 3) Normal OA Controlpxtx001-1 BMP7 Patella BV/TV 0.7566 +/− 0.011 0.6952 +/− 0.054 0.7075+/− 0.039 0.6846 +/− 0.084 TbN 7.3364 +/− 0.240  6.242 +/− 0.224 6.6528+/− 0.564 6.2122 +/− 0.169 Femur BV/TV 0.3603 +/− 0.086 0.2103 +/− 0.0850.2201 +/− 0.086 0.2245 +/− 0.066 TbN 5.7179 +/− 0.951  3.540 +/− 0.675 4.075 +/− 0.877  4.200 +/− 0.830 Tibia BV/TV 0.2773 +/− 0.047  0.157+/− 0.063 0.1469 +/− 0.057 0.1757 +/− 0.087 TbN 6.0330 +/− 0.397 4.0766+/− 1.045 4.2140 +/− 1.021 4.9095 +/− 0.719 BV/TV = The ratio of bonevolume to total volume; TbN = trabecular number; Normal = age-matcheduntreated; OA Control = experimental OA and saline; pxtx001-1 = 250 nMpeptide; BMP 7 = 500 ng. OA was induced with one injection ofmonoiodoacetate followed weekly through week 4 with infrapatellarinjection.

Following microCT analysis, joints were, decalcified, paraffin-embedded,sectioned and stained with hematoxylin and eosin (H&E) or saffranin O(for total proteoglycan content). Grading was assessed by the OARSIscale (Pritzker, K. P., Gay, S., Jimenez, S. A., Ostergaard, K.,Pelletier, J. P., Revell, P. A., Salter, D., and van den Berg, W. B.(2006) Osteoarthritis Cartilage 14, 13-29) for pathology. FIG. 23 showshistopathology results of the present experiment comparing normal (top),control (middle), and peptide inhibitor treated (bottom) stained withboth safranin-O (right), and hematoxaylin+eosin (left). Histologically,the peptide treated joints have a lower grading on the OA scale than theuntreated joints as evidenced by proteoglycan content and abnormalchondrocyte morphology. (FIGS. 24-35).

Example 11 Joint Space XRay Analysis of the OA Rat Model Treated withInhibitory Peptide

For X ray analysis, the distance from the outside of the femoral head tothe angle created by the calcaneus and the gastrocnemius tendon wasmeasured and this distance was kept consistent for each joint. Bothmedial and lateral views were taken for each limb to gather moreaccurate measurements of the joint space and to duplicate data. Wemeasured the shortest distance from the tibial cartilaginous surface tothe femoral cartilaginous surface (joint space) with the aid of a highquality metal microcaliper and clear plastic ruler. The distance betweenthe radiation source and the tissue was kept constant at 43.4 cm(distance to the film was kept at 85.9 cm). The kilovolt peak was keptat 50 kVp while the milliamp seconds were set at 1 mAs for allradiographs. We used a CMX 110 model x-ray machine by General Electric.Electron dense caliper set at 1 mm was included in each x-ray to allowfor proper measurements. (FIGS. 36 and 37)

Stride tests were also administered weekly during the course oftreatment to determine functional mobility in the animals. (FIG. 38).Briefly, rat's hind paws were inked, the animals were then timed whilethey walk a short path and the distance between hind leg strides wasmeasured (Hruska, R. E., Kennedy, S., and Silbergeld, E. K. (1979) LifeSci. 25, 171-179).

Example 12 Chondrocyte Model

Primary chondrocytes from early and late hypertrophic stage werecultured from Day 17 avian upper sternum. Late hypertrophic chondrocyteswere isolated from the core region of the avian sterna. Following 3-4hours collagenase and trypsin digestion, cells were centrifuged andfiltered through 0.45 um Nitex filter. Isolated cells were resuspendedin 1.2% alginate and forced into beaded structures with 102 mM CaCl₂ andrinsed in 0.15M NaCl for a final density of 5×10⁶ cells/ml. Alginatebead cultures were covered in 2 mls complete serum free DMEM highglucose media including 1 mM cysteine, 1 mM sodium pyruvate, 2 mML-glutamine, 50 μg/ml penicillin/streptomycin. L-ascorbic acid was addedto the culture at 30 ug/ml on day 2 and 50 ug/ml on day 5. Time coursetreatment was performed at 6, 12 or 24 hours with 10 nM, 100 nM, 250 nMPxTx001-1 or 6.5 uM commercially available MMP13 specific inhibitor(Calbiochem). Following a quick dissolution in 0.5M EDTA to releasecells from alginate cultures, total RNA was isolated through Trizolmethod and reverse-transcribed via SuperScript First-Strand SynthesisSystem (Invitrogen). cDNA samples were subjected to QuantiTech SyBrGreen(Qiagen) real time PCR. Samples were loaded into a 96 well plate intriplicate as 1 ul or 2 ul cDNA for each condition and primersrespectively. Expression of markers of chondrocyte maturation (collagentype X, MMP13 and alkaline phosphatase) was compared to an internalstandard of 18srRNA using ABI Prism 7000 sequence detection system(Applied Biosystems). Fold difference compared to untreated cultures wasgraphed using Prism Graph Pad and statistical analysis of one-way ANOVAand standard error of the mean were calculated with associated software.(FIG. 40).

Cytotoxicity was assessed by Alomar Blue Assay (Invitrogen) on primarychondrocytes and a monocyte cell line incubated for 24 hours withPxTx001-1. Absorbance was recorded at 570 nm for every hour up to 24 hrsto monitor both proliferation and metabolic activity. (FIG. 39).Toxicity in vivo was determined by blood analysis from rats that hadbeen injected with the peptide as described previously. Total cellcount, blood components and serum proteins were measured (Table 5). Allparameters measured were within normal ranges.

TABLE 5 1 injection 4 injections Normal Range Renal Function BUN 15 189-21 mg/dL Creatinine 0.4 0.3 0.05-0.65 mg/dL Liver Function AST 99 9539-111 U/L Alk Phos 272 194.5 16-302 U/L ALT 56 52 20-61 U/L TotalBilirubin 0.3 1.1 0.1-0.7 mg/dL Cholesterol 74 67.5 20-92 mg/dL CBC WBC3.6 4.55 (5.5-11.0) × 10{circumflex over ( )}3/ul RBC 5.59 6.9(5.5-10.5) × 10{circumflex over ( )}6/ul

Although certain presently preferred embodiments of the invention havebeen specifically described herein, it will be apparent to those skilledin the art to which the invention pertains that variations andmodifications of the various embodiments shown and described herein maybe made without departing from the spirit and scope of the invention.Accordingly, it is intended that the invention be limited only to theextent required by the appended claims and the applicable rules of law.

1. A compound with binding affinity for a hemopexin domain of a MMPprotein or a MMP substrate protein, wherein the compound is able tocompetitively inhibit binding of MMP to the substrate protein.
 2. Thecompound of claim 1, wherein the compound comprises a peptide or peptidemimetic.
 3. The compound of claim 2, wherein the peptide or peptidemimetic comprises a sequence greater than 50% identical to a MMPsequence.
 4. The compound of claim 2, wherein the peptide or peptidemimetic comprises a sequence greater than 50% identical to a MMPsubstrate protein sequence.
 5. The compound of claim 3, wherein the MMPis MMP13, MMP14, MMP16, MMP2, MMP9, MMP19, MMP17, MMP15, MMP20, MMP1,MMP24, MMP25, MMP3, MMP21, MMP28, MMP8, MMP12, MMP27, MMP11, MMP10. 6.The compound of claim 5, wherein the peptide or peptide mimeticcomprises a sequence selected from the group consisting of SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO: 47; SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:51; SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55; SEQ IDNO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59; SEQ ID NO: 60, SEQID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63; SEQ ID NO: 64, SEQ ID NO: 65,SEQ ID NO: 66, SEQ ID NO: 67; SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO: 71; SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ IDNO: 75; SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83; SEQ ID NO: 84,SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87; SEQ ID NO: 88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO: 91; SEQ ID NO: 92, SEQ ID NO: 93, SEQ IDNO: 94, SEQ ID NO: 95; SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQID NO: 99; SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:103; SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107; SEQID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111; SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115; SEQ ID NO: 116, SEQID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119; SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122, and SEQ ID NO:
 123. 7. The compound of claim 6,wherein the peptide or peptide mimetic comprises a sequence selectedfrom the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47.
 8. The compound of claim 7, wherein the peptide or peptide mimeticcomprises SEQ ID NO:36.
 9. The compound of claim 1, wherein the compoundinhibits the activation of TGFβ.
 10. The compound of claim 1, furthercomprising a binding domain.
 11. The compound of claim 10, wherein thebinding domain binds hyaluronic acid.
 12. A composition comprising thepeptide or peptide mimetic of claim 2 and a pharmaceutically acceptablecarrier.
 13. The composition of claim 12, wherein the peptide or peptidemimetic further comprises a binding domain with affinity for thepharmaceutically acceptable carrier.
 14. A method for inhibiting theactivation of TGFβ comprising the step of contacting a TGFβ LLC with thepeptide or peptide mimetic of claim
 1. 15. A method for treating anindication selected from the group consisting of osteoarthritis andcartilage degeneration, in a patient in need thereof comprising the stepof administering to the mammal an effective amount of the peptide orpeptide mimetic of claim
 1. 16. The method of claim 15, wherein thepeptide or peptide mimetic is administered by a step selected from thegroup consisting of subcutaneous injection, application of a cream,balm, lotion, or transdermal patch, or oral or nasal medication.
 17. Themethod of claim 16, wherein the method of application is injection intothe joint space or cartilage of the patient.
 18. An antibody havingaffinity for the compound of claim
 1. 19. The antibody of claim 18,wherein the antibody is a monoclonal or polyclonal antibody.
 20. Apeptide or peptide mimetic comprising a sequence greater than 50%identical to SEQ ID NO:36.
 21. A method of inhibiting activation of TGFβcomprising: contacting a protein with a peptide or peptide mimeticcomprising a sequence greater than 50% identical to SEQ ID NO:36. 22.The compound of claim 1, wherein the compound inhibits the cleavage ofcollagen.
 23. A compound with binding affinity for a MMP protein or aMMP substrate protein, wherein the compound is able to competitivelyinhibit binding of MMP to the substrate protein, and wherein thecompound aids in preventing cleavage of the MMP substrate protein; andwherein the compound does not have significant homology to a hemopexindomain of a MMP protein, and wherein the compound does not havesignificant affinity for a hemopexin domain of a MMP protein.